COMPOSITIONS FOR USE IN TREATMENT OF CHLAMYDIA

Abstract

This invention relates to compositions (e.g., vaccine compositions) which can be used to immunise against Chlamydia infections. The compositions comprise Chlamydia sp. antigens and antigen combinations which can be used to immunise against Chlamydia sp., used in the form of nucleic acids (e.g., mRNAs) encoding antigenic proteins or in the form of recombinant protein antigens.

Claims

1. A composition comprising: (i) a nucleic acid comprising a nucleotide sequence encoding a modified Major Outer Membrane Protein (MOMP) polypeptide, wherein the modified MOMP polypeptide has an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia trachomatis MOMP polypeptide and a non-native loop sequence between the conserved domain sequences; (ii) a nucleic acid that comprises a nucleotide sequence encoding a chimeric Chlamydia trachomatis MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia trachomatis MOMP VD sequences of different serovars of Chlamydia trachomatis; (iii) a nucleic acid that comprises a nucleotide sequence encoding a Chlamydia trachomatis CT443 polypeptide; and (iv) a nucleic acid that comprises a nucleotide sequence encoding a Chlamydia trachomatis CT584 polypeptide.

2. A nucleic acid comprising a nucleotide sequence encoding a modified Major Outer Membrane Protein (MOMP) polypeptide, wherein the modified MOMP polypeptide has an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia sp. MOMP polypeptide and a non-native loop sequence between the conserved domain sequences.

3. The nucleic acid of claim 2, wherein: 1) the modified MOMP polypeptide does not comprise a native Chlamydia sp. MOMP variable domain between the two or more conserved domain sequences; 2) the modified MOMP polypeptide comprises five conserved domain sequences of a native Chlamydia sp. MOMP polypeptide; 3) the modified MOMP polypeptide comprises a non-native loop sequence between each of the conserved domain sequences; and/or 4) the non-native loop sequence is between 3 and 30 amino acids in length.

4. The nucleic acid of claim 2, wherein: (i) a non-native loop sequence replacing VD1 comprises a sequence according to SEQ ID NO: 462 or 466; (ii) a non-native loop sequence replacing VD2 comprises a sequence according to SEQ ID NO: 463 or 467; (iii) a non-native loop sequence replacing VD3 comprises a sequence according to SEQ ID NO: 464 or 468; and/or (iv) a non-native loop sequence replacing VD4 comprises a sequence according to SEQ ID NO: 465 or 469.

5. The nucleic acid of claim 2, wherein: 1) the modified MOMP polypeptide comprises a sequence according to any one of SEQ ID NOs: 486-489 or a sequence that has at least 70%; 2) the modified MOMP polypeptide further comprises a secretion signal peptide sequence; and/or 3) the nucleic acid comprises a nucleotide sequence according to any one of SEQ ID NOs: 551-566 or a sequence that has at least 50% identity thereto.

6. A modified Major Outer Membrane Protein (MOMP) polypeptide having an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia sp. MOMP polypeptide and a non-native loop sequence between the conserved domain sequences.

7. The modified MOMP polypeptide of claim 6, wherein: 1) the modified MOMP polypeptide does not comprise a native Chlamydia sp. MOMP variable domain between the two or more conserved domain sequences; 2) the modified MOMP polypeptide comprises five conserved domain sequences of a native Chlamydia sp. MOMP polypeptide; 3) the modified MOMP polypeptide comprises a non-native loop sequence between each of the conserved domain sequences; and/or 4) the non-native loop sequence is between 3 and 30 amino acids in length.

8. The modified MOMP polypeptide of claim 6, wherein: (i) a non-native loop sequence replacing VD1 comprises a sequence according to SEQ ID NO: 462 or 466; (ii) a non-native loop sequence replacing VD2 comprises a sequence according to SEQ ID NO: 463 or 467; (iii) a non-native loop sequence replacing VD3 comprises a sequence according to SEQ ID NO: 464 or 468; and/or (iv) a non-native loop sequence replacing VD4 comprises a sequence according to SEQ ID NO: 465 or 469.

9. The modified MOMP polypeptide of claim 6, wherein the modified MOMP polypeptide comprises a sequence according to any one of SEQ ID NOs: 486-489 or a sequence that has at least 70% identity thereto.

10. A composition comprising the nucleic acid of claim 2.

11. A composition comprising the modified MOMP polypeptide of claim 6.

12-16. (canceled)

17. A method of treating or preventing a Chlamydia sp. infection in a subject, the method comprising administering the composition of claim 1 to the subject, wherein the composition further comprises a lipid nanoparticle (LNP) and the nucleic acid is encapsulated in the LNP, and wherein the infection is a C. trachomatis infection.

18. (canceled)

19. A method of treating or preventing a Chlamydia sp. infection in a subject, the method comprising administering the modified MOMP polypeptide of claim 6 to the subject, wherein the infection is a C. trachomatis infection.

20. (canceled)

21. (canceled)

22. The composition of claim 1, wherein one or more nucleic acids is a mRNA.

23. The composition of claim 22, wherein: (i) the mRNA comprises at least one 5 untranslated region (5 UTR), at least one 3 untranslated region (3 UTR), and/or at least one polyadenylation (poly(A)) sequence; and/or (ii) the mRNA is chemically modified and wherein the chemical modification consists of N1-methylpseudouridine in place of every uridine.

24. The composition of claim 1, wherein the composition further comprises a lipid nanoparticle (LNP).

25. The composition of claim 24, wherein: (i) the LNP comprises at least one cationic lipid, wherein the cationic lipid is selected from the group consisting of OF-02, cKK-E10, OF-Deg-Lin, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, SM-102, ALC-0315, ATX-126, and IM-001; and/or (ii) the LNP comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

26. The composition of claim 1, wherein: (1) the nucleic acid that comprises a nucleotide sequence encoding a Chlamydia trachomatis modified Major Outer Membrane Protein (MOMP) polypeptide is a mRNA which comprises the following structural elements: a 5 cap; a 5 untranslated region (5 UTR); a nucleotide sequence encoding the modified Major Outer Membrane Protein (MOMP) polypeptide comprising the sequence according to SEQ ID NO: 486; a 3 untranslated region (3 UTR); and a polyA tail; (2) the nucleic acid comprising a nucleotide sequence encoding the chimeric Chlamydia trachomatis MOMP VD polypeptide is a mRNA which comprises the following structural elements: a 5 cap; a 5 untranslated region (5 UTR); a nucleotide sequence encoding the chimeric Chlamydia trachomatis MOMP VD polypeptide comprising the sequence according to SEQ ID NO: 503; a 3 untranslated region (3 UTR); and a polyA tail; (3) the nucleic acid that comprises a nucleotide sequence encoding a Chlamydia trachomatis CT443 polypeptide is a mRNA which comprises the following structural elements: a 5 cap; a 5 untranslated region (5 UTR); a nucleotide sequence encoding the Chlamydia trachomatis CT443 polypeptide comprising the sequence according to SEQ ID NO 507; a 3 untranslated region (3 UTR); and a polyA tail; and (4) the nucleic acid that comprises a nucleotide sequence encoding a Chlamydia trachomatis CT584 polypeptide is a mRNA which comprises the following structural elements: a 5 cap; a 5 untranslated region (5 UTR); a nucleotide sequence encoding the Chlamydia trachomatis CT584 polypeptide comprising the sequence according to SEQ ID NO: 510; a 3 untranslated region (3 UTR); and a polyA tail; wherein the mRNA is chemically modified and wherein the chemical modification consists of N1-methylpseudouridine in place of every uridine.

27. The composition of claim 26, wherein the composition is immunogenic.

28. The nucleic acid of claim 2, wherein the Chlamydia sp. is Chlamydia trachomatis.

29. The nucleic acid of claim 3, wherein: (i) the modified MOMP polypeptide comprises all five full-length conserved domains of a native Chlamydia sp. MOMP polypeptide; (ii) the conserved domains of the modified MOMP polypeptide collectively have at least 95% sequence identity to the conserved domains of a native MOMP polypeptide; (iii) the modified MOMP polypeptide comprises four non-native loop sequences and does not comprise any native Chlamydia sp. MOMP variable domains between the conserved domain sequences; and/or (iv) the non-native loop sequence is between 4 and 20 amino acids in length.

30. The nucleic acid of claim 2, wherein the nucleic acid is a mRNA.

31. The nucleic acid of claim 30, wherein: (i) the mRNA comprises at least one 5 untranslated region (5 UTR), at least one 3 untranslated region (3 UTR), and/or at least one polyadenylation (poly(A)) sequence; and/or (ii) the mRNA is chemically modified and wherein the chemical modification consists of N1-methylpseudouridine in place of every uridine.

32. The modified MOMP polypeptide of claim 6, wherein the Chlamydia sp. is Chlamydia trachomatis.

33. The modified MOMP polypeptide of claim 7, wherein: (i) the modified MOMP polypeptide comprises all five full-length conserved domains of a native Chlamydia sp. MOMP polypeptide; (ii) the conserved domains of the modified MOMP polypeptide collectively have at least 95% sequence identity to the conserved domains of a native MOMP polypeptide; (iii) the modified MOMP polypeptide comprises four non-native loop sequences and does not comprise any native Chlamydia sp. MOMP variable domains between the conserved domain sequences; and/or (iv) the non-native loop sequence is between 4 and 20 amino acids in length.

34. The composition of claim 10, wherein the composition further comprises a lipid nanoparticle (LNP).

35. The composition of claim 34, wherein: (i) the LNP comprises at least one cationic lipid, wherein the cationic lipid is selected from the group consisting of OF-02, cKK-E10, OF-Deg-Lin, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, SM-102, ALC-0315, ATX-126, and IM-001; and/or (ii) the LNP comprises a polyethylene glycol (PEG) conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid.

36. The composition of claim 10, wherein the composition further comprises: (i) a nucleic acid that comprises a nucleotide sequence encoding a chimeric Chlamydia sp. MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia sp. MOMP VD sequences of different serovars of the Chlamydia sp.; and/or (ii) one or more of: (a) a nucleic acid that comprises a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide; (b) a nucleic acid that comprises a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide; (c) a nucleic acid that comprises a nucleotide sequence encoding a Chlamydia sp. CT600 polypeptide; and (d) a nucleic acid that comprises a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide.

37. The composition of claim 11, wherein the composition further comprises: (i) a chimeric Chlamydia sp. MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia sp. MOMP VD sequences of different serovars of the Chlamydia sp.; and/or (ii) one or more of: (a) a polypeptide comprising amino acid sequence of a Chlamydia sp. CT443 polypeptide; (b) a polypeptide comprising amino acid sequence of a Chlamydia sp. CT584 polypeptide; (c) a polypeptide comprising amino acid sequence of a Chlamydia sp. CT600 polypeptide; or (d) a polypeptide comprising amino acid sequence of a Chlamydia sp. CT812 polypeptide.

38. A method of treating or preventing a Chlamydia sp. infection in a subject, the method comprising administering the composition of claim 34 to the subject, wherein the nucleic acid is encapsulated in the LNP and wherein the infection is a C. trachomatis infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0942] FIG. 1 shows induction of cross-serovar-responsive IFN?-producing T cells in mice immunised with native SS-MOMP mRNA delivering wild type (WT) full-length MOMP (mRNA MNR MOMP E FL) or MOMP without variable domains VD2 and VD4 (mRNA UNR MOMP E DII:IV), in response to stimulation with pools of overlapping peptides of MOMP serovar D, E or F. MNR=modified mRNA (in which all uridine nucleosides were replaced with 1-methyl pseudo uridine). UNR=unmodified mRNA (containing unmodified uridine nucleosides).

[0943] FIGS. 2A-2B show induction of cross-serovar-responsive (FIG. 2A) CD4+ IFN?+ T cells and (FIG. 2B) CD8+ IFN?+ T cells in mice immunised with mRNA MNR MOMP E FL or mRNA UNR MOMP E DII:IV, in response to stimulation with pools of overlapping peptides of MOMP serovar E or F.

[0944] FIG. 3 shows reciprocal dilution IgG titres in mice immunised with LNP-formulated mRNAs encoding recombinant serovar E MOMP, assessed using plate ELISA.

[0945] FIGS. 4A-4C shows T cell responses in mice immunised with LNP-formulated mRNAs encoding recombinant serovar E MOMP in response to stimulation with (FIG. 4A) UV inactivated EBs, (FIG. 4B) rMOMP (recombinant serovar E MOMP protein), and (FIG. 4C) a pool of overlapping serovar E MOMP peptides, assessed in splenic IFN? ELISPOT.

[0946] FIGS. 5A-5B show the level of IFN? production by T cells in mice following immunisation with MOMP mRNA constructs in which one or more variable domain (VD) loops are removed, in response to stimulation with recall antigen (pools of overlapping peptides of serovar E MOMP).

[0947] FIGS. 6A-6C show the induction in mice of (FIG. 6A) IFN?-producing T cells (in response to MOMP serovar E peptide pools), (FIG. 6B) CD4+ IFN? T cells, and (FIG. 6C) CD8+ IFN? T cells following immunisation with mRNA UNR MOMP E DII:IV or recombinant serovar E MOMP protein.

[0948] FIG. 7 shows the induction of cross-serovar-responsive IFN?-producing T cells in mice following immunisation with mRNAs encoding modified MOMP polypeptide antigens in which VD loops have been removed (MOMP_P3_ssHA1 (P3), MOMP_P5_ssHA1_C2S (P5 C2S)), or full-length MOMP constructs MOMP_serE_FL_ssHA1 (FL), MOMP_serE_FL_ssHA1_C2S_Glycneg (FL C2S Glycneg). Recall antigens were overlapping peptides of MOMP serovar D, E, F, G or J.

[0949] FIG. 8 shows induction of cross-serovar-responsive IL-5-producing T cells in mice following immunisation with MOMP_P3_ssHA1 (P3), MOMP_P5_ssHA1_C2S (P5 C2S), MOMP_serE_FL_ssHA1 (FL), or MOMP_serE_FL_ssHA1_C2S_Glycneg (FL C2S Glycneg). Recall antigens were overlapping peptides of MOMP serovar D, E, F, G or J.

[0950] FIGS. 9A-9B show the induction of cross-serovar-responsive IFN?-producing T cells in mice following immunisation with mRNAs encoding modified MOMP polypeptide antigens in which VD loops have been removed (MOMP_P3_ssHA1 (P3), MOMP_P3_ssHA1_C2S (P3 C2S), MOMP_P5_ssHA1 (P5), MOMP_P5_ssHA1_C2S (P5 C2S)), or full-length MOMP constructs MOMP_serE_FL_ssHA1 (SerE FL), MOMP_serD_FL_ssHA1 (SerD FL) according to the study detailed in 7.2. Recall antigens were overlapping peptides of (FIG. 9A) MOMP serovar E or (FIG. 9B) MOMP serovar D, F, G or J.

[0951] FIGS. 10A-10B show induction in mice of (FIG. 10A) CD4+ IFN? T cell responses and (FIG. 10B) CD8+ IFN? T cell responses following immunisation with MOMP_P3_ssHA1 (P3), MOMP_P5_ssHA1_C2S (P5 C2S), MOMP_serE_FL_ssHA1 (FL), or MOMP_serE_FL_ssHA1_C2S_Glycneg (FL C2S Glycneg).

[0952] FIGS. 11A-11B show induction in mice of (FIG. 11A) CD4+ IFN? T cell responses and (FIG. 11B) CD8+ IFN? T cell responses following immunisation with MOMP_P3_ssHA1 (P3), MOMP_P3_ssHA1_C2S (P3 C2S), MOMP_P5_ssHA1 (P5), MOMP_P5_ssHA1_C2S (P5 C2S), or full-length MOMP constructs MOMP_serE_FL_ssHA1 (SerE FL), MOMP_serD_FL_ssHA1 (SerD FL) according to the study detailed in 7.2.

[0953] FIG. 12 shows the percentage of CD4+ T cells in mice that produce IFN? (IFNg), IL2 and/or TNF? (TNF?) (singularly and in the various combinations thereof), after mice are immunised with MOMP_P3_ssHA1 (P3), MOMP_P5_ssHA1_C2S (P5 C2S), MOMP_serE_FL_ssHA1 (FL), or MOMP_serE_FL_ssHA1_C2S_Glycneg (FL C2S Glycneg).

[0954] FIG. 13 shows the percentage of CD4+ T cells in mice that produce IFN? (IFNg), IL2 and/or TNF? (TNF?) (singularly and in the various combinations thereof), after mice are immunised with MOMP_P3_ssHA1 (P3), MOMP_P3_ssHA1_C2S (P3 C2S), MOMP_P5_ssHA1 (P5), MOMP_P5_ssHA1_C2S (P5 C2S), or full-length MOMP constructs MOMP_serE_FL_ssHA1 (SerE FL), MOMP_serD_FL_ssHA1 (SerD FL) according to the study detailed in 7.2.

[0955] FIG. 14 shows levels of IgG binding to recombinant MOMP (rMOMP) protein, as measured using ELISA, following immunisation of mice with MOMP_P3_ssHA1 (P3), MOMP_P3_ssHA1_C2S (P3 C2S), MOMP_P5_ssHA1 (P5), MOMP_P5_ssHA1_C2S (P5 C2S), or full-length MOMP constructs MOMP_serE_FL_ssHA1 (FL), MOMP_serD_FL_ssHA1 (SerD FL) according to the study detailed in 7.2.

[0956] FIG. 15 shows IFN? T cell responses in mice following immunisation with recombinant (r) non-MOMP polypeptides rCT584, rCT812, rCT600 or rCT443, recombinant MOMP protein (rMOMP) or mRNA encoding MOMP serovar E lacking VD loops 1-4 (mRNA MOMP ?VD1234). Pools of overlapping peptides of the respective native MOMP or non-MOMP were used as recall antigens.

[0957] FIG. 16 shows IFN? T cell responses in mice following immunisation with non-MOMP fragment mRNA constructs (CT812: 1-1530, CT812: 1-761, Nan96-SS:CT812: 32-761, CT600: 1-188, CT443: 1-576, Nan96-SS:CT443: 32-576), or with mRNA encoding MOMP serovar E lacking VD loops 1-4 (MOMP ?VD1234). Pools of overlapping peptides of the respective native MOMP or non-MOMP were used as recall antigens.

[0958] FIG. 17 shows IFN? T cell responses in mice following immunisation with mRNA encoding non-MOMP Ct antigens CT443 (Nan96-SS:CT443: 32-576), CT584 (Nan96HA-SS:CT584:2-183), CT600 (Nan96HA-SS:CT600:2-188) or CT812 (Nan96-SS:CT812:32-761), or with recombinant MOMP serovar E protein, in response to stimulation with respective overlapping peptide pools. Responses are shown for splenocytes collected at days eleven (D11) and fourteen (D14) post-boost.

[0959] FIGS. 18A-18B show induction in mice of (FIG. 18A) CD4+ IFN? T cell responses and (FIG. 18B) CD8+ IFN? T cell responses following immunisation with mRNA encoding non-MOMP Ct antigens CT443 (Nan96-SS:CT443: 32-576), CT584 (Nan96HA-SS:CT584:2-183), CT600 (Nan96HA-SS:CT600:2-188) or CT812 (Nan96-SS:CT812:32-761), or with recombinant MOMP serovar E protein.

[0960] FIG. 19 shows antigen-specific IgG levels, as measured using ELISA, following immunisation with mRNA encoding non-MOMP Ct antigens CT443_ssHA1 (CT443), CT443_ssHA1_GlycNeg (CT443_GN), CT584_ssHA1 (CT584), CT584_ssHA1_Glycneg (CT584_GN), CT584_ssHA1_C2S (CT584_C2S), CT584_ssHA1_Glycneg_C2S (CT584_GN_C2S), CT600_trunc_ssHA1 (CT600 trunc), CT600_trunc_ssHA1_Glycneg (CT600_trunc_GN), CT812_pass-domain_ssHA1 (CT812_pass-dom), CT812_pass-domain_ssHA1_C2S (CT812_pass-dom_C2S), CT812_ext-pass-domain_ssHA1 (CT812_ext-pass-dom), CT812_ext-pass-domain_ssHA1_C2S (CT812_ext-pass-dom_C2S).

[0961] FIG. 20 shows induction of IFN?-producing T cells in mice following immunisation with mRNA encoding non-MOMP Ct antigens CT443_ssHA1 (CT443), CT584_ssHA1_GlycNeg (CT584_GlycNeg), or CT600_trunc_ssHA1_Glycneg (CT600 GlycNeg), or with mRNA encoding recombinant MOMP serovar E protein as a control.

[0962] FIGS. 21A-21B show induction in mice of (FIG. 21A) CD4+ IFN? T cell responses and (FIG. 21B) CD8+ IFN? T cell responses, following immunisation with mRNA encoding non-MOMP Ct antigens CT443_ssHA1 (CT443), CT584_ssHA1_GlycNeg (CT584_GlycNeg), or CT600_trunc_ssHA1_Glycneg (CT600_GlycNeg), or with mRNA encoding recombinant MOMP serovar E protein as a control.

[0963] FIGS. 22A-22B show the percentage of (FIG. 22A) CD4+ T cells and (FIG. 22B) CD8+ T cells in mice producing each of IFN? (IFNg), IL2, and/or TNF? (TNFa) (singularly and in the various combinations thereof), after mice are immunised with mRNA encoding non-MOMP Ct antigens CT443_ssHA1 (CT443), CT584_ssHA1_GlycNeg (CT584_GlycNeg), or CT600_trunc_ssHA1_Glycneg (CT600_GlycNeg), or with mRNA encoding recombinant MOMP serovar E protein as a control.

[0964] FIG. 23 shows antigen-specific IgG levels, as measured using ELISA, following immunisation of mice with mRNA encoding non-MOMP Ct antigens CT443_ssHA1 (CT443 mRNA) CT584_ssHA1_GlycNeg (CT584 mRNA), CT600_trunc_ssHA1_Glycneg (CT600 mRNA) or with one of recombinant proteins (rProt) CT584, CT600 and CT443.

[0965] FIG. 24 shows the level of IgG binding to EBs of serovar E, as measured using ELISA, following immunisation of mice with mRNA encoding non-MOMP Ct antigens CT443_ssHA1 (CT443), CT443_ssHA1_GlycNeg (CT443_GN), CT584_ssHA1 (CT584), CT584_ssHA1_GlycNeg (CT584_GN), CT584_ssHA1_C2S (CT584_C2S), CT600_trunc_ssHA1_Glycneg (CT600_trunc), CT600_trunc_ssHA1_Glycneg (CT600_trunc_GN), CT812_pass-domain_ssHA1 (CT812_pass-dom), CT812_pass-domain_ssHA1_C2S (CT812_pass-dom_CS2), CT812_ext-pass-domain_ssHA1 (CT812_ext-pass-dom), or CT812_ext-pass-domain_ssHA1_C2S (CT812_ext-pass-dom_C2S).

[0966] FIG. 25 shows the level of IgG binding to EBs of serovar D, as measured using ELISA, following immunisation of mice with mRNA encoding non-MOMP Ct antigens as described above in the legend of FIG. 24.

[0967] FIG. 26 shows the level of IgG binding to EBs of serovar G, as measured using ELISA, following immunisation of mice with mRNA encoding non-MOMP Ct antigens as described above in the legend of FIG. 24.

[0968] FIG. 27 shows antigen-specific IgG levels, as measured using ELISA, following immunisation of mice with mRNA encoding various chimeric MOMP VD polypeptides, with mRNA encoding full-length MOMP polypeptides, or with rMOMP protein.

[0969] FIG. 28 shows the level of IgG binding to EBs of serovar E, as measured using ELISA, following immunisation of mice with mRNA encoding various chimeric MOMP VD polypeptides, with mRNA encoding full-length MOMP polypeptides, or with rMOMP protein.

[0970] FIG. 29 shows the level of IgG binding to EBs of serovar D, as measured using ELISA, following immunisation of mice with mRNA encoding various chimeric MOMP VD polypeptides, with mRNA encoding full-length MOMP polypeptides, or with rMOMP protein.

[0971] FIG. 30 shows the level of IgG binding to EBs of serovar G, as measured using ELISA, following immunisation of mice with mRNA encoding various chimeric MOMP VD polypeptides, with mRNA encoding full-length MOMP polypeptides, or with rMOMP protein.

[0972] FIGS. 31A-31C show induction in mice of the (FIG. 31A) CD4+ IFN? T cell response, (FIG. 31B) CD8+ IFN? T cell response, and (FIG. 31C) CD4+ IFN?+IL2+TNF?+ cell population, following immunisation with mRNA encoding MOMP_P3_ssHA1 (P3) in two different LNP formulations or MOMP_VDcomb2-extS_ssHA1_Glycneg (C2-S-GN). Empty LNPs served as controls.

[0973] FIGS. 32A-32B show (FIG. 32A) the level of antigen-specific IgG against recombinant MOMP (rMOMP) of serovar E and (FIG. 32B) the level of IgG binding to EBs of serovar E, as measured using ELISA, following immunisation of mice with mRNA encoding MOMP_VDcomb2-extS_ssHA1_Glycneg (C2-S-GN) in two different LNP formulations. Empty LNPs and pre-immune sera (pre-immun) served as controls.

[0974] FIGS. 33A-33B show induction in mice of (FIG. 33A) the CD4+ IFN? T cell response and (FIG. 33B) the percentage of CD4+ T cells in mice that produce IFN? (IFN?), IL2 and/or TNF? (singularly and in the various combinations thereof), following immunisation with the multivalent composition as described in Example 12, formulated in two different LNP formulations and at three different doses. Empty LNPs corresponding to the amount of LNP used for the doses of 14.4 ?g and 4.8 ?g mRNA (equiv. LNP) served as controls.

[0975] FIGS. 34A-34B show induction in mice of (FIG. 34A) the CD8+ IFN? T cell response and (FIG. 34B) the percentage of CD8+ T cells in mice that produce IFN? (IFN?), IL2 and/or TNF? (singularly and in the various combinations thereof), following immunisation with a multivalent composition as described in Example 12, formulated in two different LNP formulations and at three different doses. Empty LNPs corresponding to the amount of LNP used for the doses of 14.4 ?g and 4.8 ?g mRNA (equiv. LNP) served as controls.

[0976] FIGS. 35A-35B show (FIG. 35A) the level of antigen-specific IgG against recombinant MOMP (rMOMP) of serovar E and (FIG. 35B) the level of IgG binding to EBs of serovar E, as measured using ELISA, following immunisation of mice with a multivalent composition as described in Example 12, in two different LNP formulations and at three different doses. IgG induced by empty LNPs corresponding to the amount of LNP used for the doses of 14.4 ?g and 4.8 ?g mRNA (equiv. LNP) and in pre-immune sera (pre-immun) are shown as controls.

[0977] FIGS. 36A-36B show the level of antigen-specific IgG against the (FIG. 36A) CT443 protein and (FIG. 36B) CT584 protein, as measured using ELISA, following immunisation of mice with a multivalent composition as described in Example 12, in two different LNP formulations and at three different doses. IgG induced by empty LNPs corresponding to the amount of LNP used for the doses of 14.4 ?g and 4.8 ?g mRNA (equiv. LNP) and in pre-immune sera (pre-immun) are shown as controls.

[0978] FIGS. 37A-37B show levels of IgG binding to EBs of serovar E, as measured using ELISA, following immunisation of mice with mRNA encoding monovalent polypeptides or a multivalent composition as described in Example 13, formulated in either (FIG. 37A) Lipid D or (FIG. 37B) Lipid G. *** corresponds to a p-value of p<0.001.

[0979] FIGS. 38A-38B show levels of IgG binding to EBs of serovar G, as measured using ELISA, following immunisation of mice with mRNA encoding monovalent polypeptides or a multivalent composition as described in Example 13, formulated in either (FIG. 38A) Lipid D or (FIG. 38B) Lipid G. *** corresponds to a p-value of p<0.001.

[0980] FIGS. 39A-39F show levels of IgG binding to recombinant proteins (FIG. 39A and FIG. 39B: MOMP; FIG. 39C and FIG. 39D: CT443; FIG. 39E and FIG. 39F: CT584), as measured using ELISA, following immunisation of mice with mRNA encoding monovalent polypeptides or a multivalent composition as described in Example 13, formulated in either (FIG. 39A, FIG. 39C, FIG. 39E) Lipid D or (FIG. 39B, FIG. 39D, FIG. 39F) Lipid G.

MODES FOR CARRYING OUT THE INVENTION

Example 1Design of C. trachomatis (Ct) Antigen Constructs

[0981] Design of T cell constructs (modified MOMP polypeptide constructs) mRNA constructs encoding modified MOMP polypeptide antigens were prepared with the aim of eliciting MOMP-specific T cell responses, particularly MOMP specific IFN?+CD4+ T cellsi.e. MOMP T cell constructs.

[0982] MOMP T-cell constructs MOMP_P3_ssHA1 (P3) and MOMP_P5_ssHA1 (P5) were designed using a structure-based strategy in order to replace VD1-4 regions (which contain B-cell epitopes).

[0983] As a basis for the structure guided design, Alphafold2 (Deepmind) was used to create a 3D model of the MOMP protein. The resulting MOMP model structure can be described as a beta-barrel made up of 10 antiparallel beta strands. The VD1-4 regions are situated in loops linking individual beta-strands, all located on the same side of the barrel structure.

[0984] In order to design replacement sequences for the VD1-4 loops, each VD sequence was mapped onto the MOMP model structure. The distance between the start and end of each VD region was measured, and the approximate peptide sequence length needed to cover this distance between the start and end points of each VD was estimated.

[0985] The Crosslink Proteins (CS) protocol available through Maestro (v12.8, Schrodinger) was used to build peptide sequences of diverse lengths and amino-acid composition on the MOMP model structure, in order to replace the VD regions. After energy minimization and visual inspection of resulting MOMP structures, a shortlist of 6 MOMP VD-replaced sequences were selected for further analysis.

[0986] In order to avoid issues related to human cross reactivity for the newly designed VD-replaced MOMP sequences, sequence motifs of 8 or more amino acids in the VD-replaced MOMP sequences, which are also present in the human proteome, were excluded from potential designs.

[0987] Finally, a VD1-4-replaced MOMP sequence was selected in which the VD replacement peptides/loops are predicted to have minimal impact on the overall MOMP beta-barrel structure, and which has no sequence motif (8 or more amino acids) in common with sequences present in the human proteome.

[0988] The resulting T-cell constructs MOMP_P3_ssHA1 (P3) and MOMP_P5_ssHA1 (P5) are given by SEQ ID NOs: 53 and 57, respectively.

Design of B Cell Constructs (Chimeric MOMP VD Polypeptide Constructs)

[0989] Further mRNA constructs encoding chimeric MOMP VD polypeptide antigens were prepared with the aim of eliciting MOMP-specific antibody (B cell) responsesi.e. MOMP B cell constructs.

[0990] The exemplary B cell constructs are chimeras which were designed by combining variable domains (VD) from 4 serovars of C. trachomatis (SerD, SerE, SerF & SerG). A chimeric antigen was obtained which combines copies of each of the 4 variable domains (VD1 to VD4) from different serovars, designed in this way in order to be able to induce a cross-serovar immune (antibody) response. A protein sequence comparison of the VD loops across serovars (SEQ ID NOs: 5 to 20) indicated that: [0991] VD1 loops of SerD and SerE are highly similar, and VD1 loops of SerF and SerG are identical [0992] VD2 loops of SerD and SerE are highly similar, and VD2 loops of SerF and SerG are highly similar [0993] VD3 loops of SerD and SerF are identical, and VD3 loops of SerF and SerG are highly similar [0994] VD4 loops of SerD and SerE are highly similar, and VD4 loops of SerF and SerG are highly similar

[0995] It was hypothesized that highly similar or identical sequences of VD loops between two serovars might be able to contribute to a cross-serovar response at least between those two serovars. Therefore, the designed chimeras contained two of each of VD1, VD2 and VD4 (i.e., 1 VD from D or E+1 VD for F or G, for each of the VD loops). Due to a putative homology with a human protein the VD3 from SerE was excluded from the designs and only one VD3 (from SerG or SerD/F*) was included.

[0996] To maintain the VD loops in a conformation which is structurally similar to that of the VD loops within native MOMP, some of the flanking residues either side of the VD loops were included in the designed constructs. Two construct groups with different lengths of flanking sequences either side of the VD loops were designed (extP and extS).

[0997] Two construct groups with different combinations of VD loops were also designed (VDcomb1 and VDcomb2). [0998] VDcomb1 contains the following VD loops:
VD1 (serD)-VD1 (serF/G*)-VD2 (serE)-VD2 (serF)-VD3 (serG)-VD4 (serE)-VD4 (serG) [0999] VDcomb2 contains the following VD loops:
VD1 (serE)-VD1 (serF/G*)-VD2 (serD)-VD2 (serG)-VD3 (serD/F*)-VD4 (serD)-VD4 (serF)
* the sequences for this particular VD from these serovars are identical

[1000] The resulting B cell constructs are given by protein sequences according to SEQ ID NO: 61-92.

Example 2Expression of mRNA Constructs

[1001] mRNA constructs encoding a selection of C. trachomatis (Ct) antigens were provided and expressed in HeLa cells as described in the Methods section.

Results

[1002] The following constructs were expressed and detected in lysate and/or supernatant, as indicated in Table 4 below. Expression in the supernatant indicated that the protein was secreted.

[1003] ssHA1=signal peptide sequence of SEQ ID NO:187; ssHA2=signal peptide sequence of SEQ ID NO:188; TMB1=transmembrane domain sequence of SEQ ID NO: 189; TMB2=transmembrane domain sequence of SEQ ID NO: 190; FL=full-length protein; C2S=variant containing cysteine to serine mutations which are designed to remove any disulphide bridges; GlycNeg=variant containing point mutations designed to prevent glycosylation; P3 and P5=MOMP variants (T cell constructs) in which the variable loops (VDs) have been replaced using a structure-based strategy according to the design strategy described in Example 1; VDcomb=chimeric MOMP variants (B cell constructs) in which variable loops (VDs) from different serovars are combined according to the design strategy described in Example 1; extP and extS=VDcomb construct which have different lengths of flanking sequences either side of the VD included (see Example 1 for design strategy); trunc=a truncated version of the relevant protein; CT82_pass-domain=CT812 fragment corresponding to amino acid residues 52-1003 of CT812 (SEQ ID NO: 515); CT812_ext_pass-domain=CT812 fragment corresponding to amino acid residues 52-1179 of CT812 (SEQ ID NO: 515); HPXn-n_HPXn-n=constructs in which sequence motifs of 8 or more amino-acids, which are also present in the human proteome, were mutated in order to avoid human cross reactivity; N.D.=not determined.

TABLE-US-00009 TABLE 4 mRNA constructs expressed in HeLa cells SEQ Lysate Supernatant ID NO Construct Name Expression Expression Full Length MOMP 879 MOMP_serD_FL_ssHA1 +++ + 294 MOMP_serD_FL_ssHA1 +++ + 297 MOMP_serD_FL_ssHA1_Glycneg +++ + 298 MOMP_serD_FL_ssHA1_Glycneg +++ + 873 MOMP_serD_FL_ssHA1_C2S_Glycneg +++ +++ 875 MOMP_serE_FL_ssHA1 +++ ++ 876 MOMP_serE_ssHA1_C2S_Glycneg +++ + 880 MOMP_serF_FL_ssHA1 +++ + 326 MOMP_serF_FL_ssHA1 +++ + (brighter) 338 MOMP_serF_FL_ssHA2_Glycneg +++ +++ 881 MOMP_serG_FL_ssHA1 +++ + 342 MOMP_serG_FL_ssHA1 +++ + (brighter) 345 MOMP_serG_FL_ssHA1_Glycneg + +++ T Cell Constructs 213 MOMP_P3_ssHA1 + not detected (Brighter) 218 MOMP_P3_ssHA1_C2S + + (Brighter) 219 MOMP_P3_ssHA2_C2S ++ ++ 222 MOMP_P5_ssHA1 +++ + 225 MOMP_P5_ssHA1_C2S + + B Cell Constructs 229 MOMP_VDcomb1-extP_ssHA1 +++ + 863 MOMP_VDcomb1-extP_ssHA1 +++ + 877 MOMP_VDcomb1-extP_ssHA1_Glycneg +++ ++ 232 MOMP_VDcomb1-extP_ssHA1_Glycneg +++ +++ 233 MOMP_VDcomb1-extP_ssHA1_TMB1 + N.D. 234 MOMP_VDcomb1-extP_ssHA1_TMB1 + N.D. 235 MOMP_VDcomb1-extP_ssHA1_TMB1_Glycneg ++ N.D. 865 MOMP_VDcomb1-extP_ssHA1_TMB1_Glycneg ++ N.D. 237 MOMP_VDcomb1-extP_ssHA2 +++ + 238 MOMP_VDcomb1-extP_ssHA2 +++ + 239 MOMP_VDcomb1-extP_ssHA2_Glycneg +++ ++ 242 MOMP_VDcomb1-extP_ssHA2_TMB2 + N.D. 243 MOMP_VDcomb1-extP_ssHA2_TMB2_Glycneg ++ N.D. 244 MOMP_VDcomb1-extP_ssHA2_TMB2_Glycneg ++ N.D. 245 MOMP_VD-comb1-extS_ssHA1 +++ not detected 864 MOMP_VD-comb1-extS_ssHA1 +++ +++ 878 MOMP_VD-comb1-extS_ssHA1_Glycneg +++ +++ 248 MOMP_VD-comb1-extS_ssHA1_Glycneg +++ +++ 249 MOMP_VD-comb1-extS_ssHA1_TMB1 ++ N.D. 251 MOMP_VD-comb1-extS_ssHA1_TMB1_Glycneg ++ N.D. 866 MOMP_VD-comb1-extS_ssHA1_TMB1_Glycneg ++ N.D. 253 MOMP_VD-comb1-extS_ssHA2 +++ +++ 254 MOMP_VD-comb1-extS_ssHA2 +++ +++ 255 MOMP_VD-comb1-extS_ssHA2_Glycneg +++ +++ 256 MOMP_VD-comb1-extS_ssHA2_Glycneg +++ +++ 257 MOMP_VD-comb1-extS_ssHA2_TMB2 ++ N.D. 258 MOMP_VD-comb1-extS_ssHA2_TMB2 ++ N.D. 259 MOMP_VD-comb1-extS_ssHA2_TMB2_Glycneg ++ N.D. 260 MOMP_VD-comb1-extS_ssHA2_TMB2_Glycneg ++ N.D. 261 MOMP_VDcomb2-extP_ssHA1 ++ + 262 MOMP_VDcomb2-extP_ssHA1 ++ + 868 MOMP_VDcomb2-extP_ssHA1_Glycneg +++ ++ 264 MOMP_VDcomb2-extP_ssHA1_Glycneg +++ + 265 MOMP_VDcomb2-extP_ssHA1_TMB1 + N.D. 266 MOMP_VDcomb2-extP_ssHA1_TMB1 + N.D. 871 MOMP_VDcomb2-extP_ssHA1_TMB1_Glycneg ++ N.D. 268 MOMP_VDcomb2-extP_ssHA1_TMB1_Glycneg + N.D. 269 MOMP_VDcomb2-extP_ssHA2 ++ + 270 MOMP_VDcomb2-extP_ssHA2 ++ + 869 MOMP_VDcomb2-extP_ssHA2_Glycneg +++ ++ 272 MOMP_VDcomb2-extP_ssHA2_Glycneg +++ ++ 273 MOMP_VDcomb2-extP_ssHA2_TMB2 + N.D. 274 MOMP_VDcomb2-extP_ssHA2_TMB2 + N.D. 867 MOMP_VDcomb2-extP_ssHA2_TMB2_Glycneg + N.D. 276 MOMP_VDcomb2-extP_ssHA2_TMB2_Glycneg + N.D. 277 MOMP_VDcomb2-extS_ssHA1 +++ +++ 278 MOMP_VDcomb2-extS_ssHA1 +++ +++ 870 MOMP_VDcomb2-extS_ssHA1_Glycneg +++ +++ 280 MOMP_VDcomb2-extS_ssHA1_Glycneg +++ +++ 872 MOMP_VDcomb2-extS_ssHA1_TMB1 + N.D. 282 MOMP_VDcomb2-extS_ssHA1_TMB1 + N.D. 283 MOMP_VDcomb2-extS_ssHA1_TMB1_Glycneg ++ N.D. 285 MOMP_VDcomb2-extS_ssHA2 +++ +++ 287 MOMP_VDcomb2-extS_ssHA2_Glycneg +++ +++ 290 MOMP_VDcomb2-extS_ssHA2_TMB2 ++ N.D. 291 MOMP_VDcomb2-extS_ssHA2_TMB2_Glycneg ++ N.D. 292 MOMP_VDcomb2-extS_ssHA2_TMB2_Glycneg ++ N.D. Non-MOMP Constructs 369 CT443_ssHA1 PolyA + not detected (band size is at albumin band) 370 CT443_ssHA1 PolyA + not detected (band size is at albumin band) 371 CT443_ssHA1_Glycneg PolyA + not detected (band size is at albumin band) 372 CT443_ssHA1_Glycneg PolyA + not detected (band size is at albumin band) 373 CT584_ssHA1 PolyA + + 374 CT584_ssHA1 PolyA +++ +++ 375 CT584_ssHA1_C2S PolyA ++ ++ 376 CT584_ssHA1_C2S PolyA ++ ++ 377 CT584_ssHA1_Glycneg PolyA +++ +++ 378 CT584_ssHA1_Glycneg PolyA +++ +++ 379 CT584_ssHA1_Glycneg_C2S PolyA ++ ++ 380 CT584_ssHA1_Glycneg_C2S PolyA ++ ++ 381 CT600_trunc_ssHA1 +++ +++ 382 CT600_trunc_ssHA1 +++ ++ 383 CT600_trunc_ssHA1_Glycneg +++ +++ 384 CT600_trunc_ssHA1_Glycneg +++ +++ 385 CT812_pass-domain_ssHA1 ++ not detected (brighter) 386 CT812_pass-domain_ssHA1 ++ not detected 387 CT812_pass-domain_ssHA1_C2S ++ not detected 388 CT812_pass-domain_ssHA1_C2S ++ not detected 389 CT812_ext-pass-domain_ssHA1 + not detected 390 CT812_ext-pass-domain_ssHA1 + not detected 391 CT812_ext-pass-domain_ssHA1_C2S PolyA + not detected 392 CT812_ext-pass-domain_ssHA1_C2S PolyA + not detected 393 CT812_pass-domain_ssHA1_HPX1-2_HPX2-1 CO1 +++ + 394 CT812_pass-domain_ssHA1_HPX1-2_HPX2-1 CO2 +++ +/? 395 CT812_pass-domain_ssHA1_HPX1-2_HPX2-1_C2S CO1 +++ +/? 396 CT812_pass-domain_ssHA1_HPX1-2_HPX2-1_C2S CO2 +++ +/? 397 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-3_C2S CO1 +++ + 398 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-3_C2S CO2 +++ + 399 CT812_pass-domain_ssHA1_HPX1-1_HPX2-1 CO1 +++ + 400 CT812_pass-domain_ssHA1_HPX1-1_HPX2-1 CO2 +++ +/? 401 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-3 CO1 +++ + 402 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-3 CO2 +++ + 403 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-1 CO1 +++ + 404 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-1 CO2 +++ + 405 CT812_pass-domain_ssHA1_HPX1-2_HPX2-3 CO1 +++ + 406 CT812_pass-domain_ssHA1_HPX1-2_HPX2-3 CO2 +++ + 407 CT812_pass-domain_ssHA1_HPX1-2_HPX2-3_C2S CO1 +++ +/? 408 CT812_pass-domain_ssHA1_HPX1-2_HPX2-3_C2S CO2 +++ +/? 409 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-1_C2S CO1 +++ + 410 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-1_C2S CO2 +++ + 411 CT812_pass-domain_ssHA1_HPX1-1_HPX2-3 CO1 +++ + 412 CT812_pass-domain_ssHA1_HPX1-1_HPX2-3 CO2 +++ +/? 413 CT812_pass-domain_ssHA1_HPX1-1_HPX2-1_C2S CO1 +++ +/? 414 CT812_pass-domain_ssHA1_HPX1-1_HPX2-1_C2S CO2 +++ +/? 415 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-3_C2S CO1 +++ + 416 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-3_C2S CO2 +++ + 417 CT812_pass-domain_ssHA1_HPX1-1_HPX2-3_C2S CO1 +++ + 418 CT812_pass-domain_ssHA1_HPX1-1_HPX2-3_C2S CO2 +++ +/? 419 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-3 CO1 +++ + 420 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-3 CO2 +++ + 421 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-1 CO1 +++ + 422 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-1 CO2 +++ + 423 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-1_C2S CO1 +++ + 424 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-1_C2S CO2 +++ + Control Constructs 862 Hirep2_ssHA1 +++ ++ 202 Hirep2_ssHA1 +++ + 365 CTH522_patent_ssHA1 [CO1] PolyA +++ + 366 CTH522_patent_ssHA1 [CO2] PolyA +++ + 367 CTH522_patent_ssHA1_Glycneg [CO1] PolyA +++ +

Methods

[1004] HeLa cells were plated in 12-well plates at 0.15 million cells/well in 1 mL EMEM+10% FBS. Cells were transfected the next day with 1 ?g/well Chlamydia mRNA constructs with lipofectamine 2000.

Preparation of Lysates

[1005] Cells were harvested the 22-24 hours later and lysed in 250 ?L per well of CelLytic M+1?HALT. Lysates were incubated on ice for 10 minutes then cleared in a microcentrifuge at max speed for 10 min at 4? C. 15 ?L Lysate was combined with 5 ?L NuPAGE LDS Sample buffer with reducing agent. Reduced samples were incubated at 85? C. for 5 minutes.

Preparation of Supernatant Samples

[1006] Supernatants were harvested the 22-24 hours after transfection. 15 ?L supernatant was combined with 5 ?L NuPAGE LDS Sample buffer with reducing agent. Reduced samples were incubated at 85? C. for 5 minutes.

Analysis

[1007] The resulting lysate or supernatant samples were run on 8-16% Gradient SDS-PAGE at 185V for 60 minutes. Protein was transferred to nitrocellulose membrane. Blots were blocked with Intercept blocking buffer overnight at 4? C. Blots were stained with anti-Chlamydia trachomatis MOMP polyclonal antibody (for full-length MOMP constructs, T cell constructs, B cell constructs and control constructs), anti-CT443 polyclonal sera (for CT443 constructs), anti-CT584 polyclonal sera (for CT584 constructs), anti-CT600 polyclonal sera (for CT600 constructs) or anti-CT812 polyclonal sera (for CT812 constructs). Antibody staining was done in Intercept Blocking buffer+0.2% Tween 20 for 2 hrs at room temperature (RT). Blots were washed with TBST 3?5 minutes. Blots stained with anti-Chlamydia trachomatis MOMP polyclonal antibody were then stained with donkey anti-goat 800 secondary antibody in Intercept Blocking buffer+0.2% Tween 20 for 1 hr at RT. Blots stained with anti-CT443, anti-CT584, anti-CT600 or anti-CT812 polyclonal sera were then stained with donkey anti-rabbit 800 secondary antibody in Intercept Blocking buffer+0.2% Tween 20 for 1 hr at room temperature. Blots were washed with TBST 4?5 minutes. Blots were scanned on Licor odyssey.

Example 3Recombinant Protein Expression in E. coli

[1008] DNA constructs encoding Ct antigens were cloned into a T7 promoter-based expression vector for expression in E. coli using standard methods. E. coli (BL21 DE3) were transformed with the vector. Protein expression was induced with IPTG. The constructs were purified from inclusion bodies using nickel chelation chromatography, followed by protein refolding using the buffers outlined in Table 5.

TABLE-US-00010 TABLE 5 Constructs and composition of refolding buffers used for each construct Construct Refolding buffer MOMP_P5_6his_coli 50 mM Tris-HCl, 150 mM (encoding protein of SEQ NaCl, 10% Glycerol, 0.2% ID NO: 133) SDS, pH 8.0 MOMP_SerE_FL_Wt Refolding buffer containing (encoding protein of SEQ 0.1% NLS detergent ID NO: 134) MOMP_serE_FL_C2S.sub. 20 mM Tris, 150 mM NaCl, 6his_coli (encoding protein 0.1% NLS of SEQ ID NO: 136) CT443_6his_coli (encoding PBS, 10% Glycerol, 500 mM protein of SEQ ID NO: 143) NaCl, pH 7.4 6his_CT584_coli (encoding 20 mM Tris, 150 mM NaCl, protein of SEQ ID NO: 145) 0.1% NLS 6his_CT600_coli (encoding PBS, 0.2% NLS, pH 7.4 protein of SEQ ID NO: 146) CT812_ext-pass-domain.sub. PBS, 10% Glycerol, 0.2% 6his_coli (encoding protein Sodium Lauroyl Sarcosine, of SEQ ID NO: 147) pH 7.4

[1009] The purified constructs were analysed using SDS-PAGE gel electrophoresis under reducing conditions. Western blotting using an anti-His tag antibody was performed for all the constructs except MOMP_serE_FL, and in each case detected the expressed product at the expected molecular weight on the blot. SDS-PAGE under non-reducing conditions indicated that MOMP_serE_FL adopts a trimeric form. Construct stability and conformation were assessed by differential scanning calorimetry (DSC), and secondary structure was assessed using circular dichroism (CD).

Example 4Immunisation with MOMP mRNA Constructs Elicits Cross-Serovar T Cell Responses in Mice

[1010] Selected mRNA constructs encoding a native SS-MOMP delivering wild type (WT) full-length MOMP (mRNA MNR MOMP E FL; encoding protein of SEQ ID NO: 155) and MOMP without variable domains VD2 and VD4 (mRNA UNR MOMP E DII:IV; encoding protein of SEQ ID NO: 157) were tested for their ability to elicit T cell responses. SS-MOMP=native secretion signal sequence of Ct MOMP (SEQ ID NO: 192). As used herein, MNR=modified mRNA (in which all uridine nucleosides were replaced with 1-methyl pseudo uridine). UNR=unmodified mRNA (containing unmodified uridine nucleosides). As used herein in the examples, modified mRNAs (or MNRs) have the nucleic acid sequence as indicated by the SEQ ID NO wherein all uridine nucleosides have been replaced with 1-methylpseudouridine.

[1011] In the mRNA UNR MOMP E DII:IV construct, nucleic acid sequences encoding variable loops VD2 and VD4 were replaced with sequences encoding non-native loop motifs containing exclusively glycine residues, in order to remove any immunogenic epitopes contained within the loops.

[1012] Mice received two immunisations of the SS-MOMP constructs (2 ?g dose), formulated with the LNP OF-02, given by IM route, at 0 and 3 weeks (W0 and W3). Two additional groups were injected with the mRNA MNR MOMP E FL construct according to the same methods but at 0.5 ?g and 0.125 ?g doses, respectively. 10 mice per group were immunised in each group. Studies included two negative controls: PBS (5 mice) or empty LNP OF-02 (10 mice). Splenocytes were collected fourteen days post-boost. The T specific cellular response was assessed by ELISPOT/FluoroSpot and by Intra cellular cell staining (ICCS).

[1013] LNP OF-02 (Lipid A)=cationic lipid OF-02 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%

Ability to Elicit IFN?-Producing T Cells (FluoroSpot)

[1014] First, ability to elicit IFN?-producing T cells was tested using FluoroSpot. For the FluoroSpot assay, spleen cells (0.2?10.sup.6 per well) were plated in 200 ?l RPMI complete together with and without MOMP peptide pools from serovar D, E and F in presence of IL2 (Roche) in triplicate cultures each. The overlapping peptide pools used herein for restimulation consisted of 15-residue peptides covering the entire native protein sequence of interest with 11-residue overlaps between consecutive peptides. Subsequently, plates were incubated at 37? C., 5% CO.sub.2 for 20 h. After washing with PBS, plates were incubated with detection Ab anti-IFN? (mAb R4-6A2-BAM) for 2 h at RT. Image analysis of FluoroSpot assays was performed on Microvision Reader.

[1015] To assess the antigen specific response in an ELISPOT/FluoroSpot assay as described herein the number of cytokine producing spots are assessed after either incubation with a specific antigen (peptide, protein, whole bacteria) or no antigen. The average number of cytokine forming spots detected after an incubation with no antigen is considered the assay negative control and this typically very small number of spots is subtracted from the number of cytokine forming spots formed after the different antigen stimulations. This is how the ELISPOT/FluoroSpot data is normalized. Thus, in an ELISPOT/FluoroSpot assay as described herein cells are plated together with and without peptide pools, assessing both the response to antigen stimulation (with peptide pools) and no antigen stimulation (without peptide pools). The without peptide data are used to normalize the antigen specific data shown in the corresponding Figures.

[1016] As shown in FIG. 1, both mRNA MNR MOMP E FL and mRNA UNR MOMP E DII:IV (2 ?g) were able to induce a comparable level of cross-serovar-responsive IFN?-producing T cells, suggesting that the T cell response is maintained despite removal of VD loops. Responses were dose-dependent, with little observable difference between 2 ?g and 0.5 ?g. A 0.125 ?g dose was therefore used in later studies comparing mRNA constructs.

Ability to Elicit CD4+ IFN?+ and CD8+ IFN?+ T Cell Responses (ICCS)

[1017] The MOMP mRNA constructs were also tested for their ability to elicit CD4+ IFN?+ T cells and CD8+ IFN?+ T cells in mice that are reactive across various serovars.

[1018] For ICCS, isolated lymphocytes were activated with specific serovar E- and F MOMP peptide pools (as described above) at 1?10.sup.6 cells/well in 96-well plates and cultured for 6 h in 5% CO.sub.2 at 37? C. with BFA in order to block cytokine secretion. Activated cells were harvested for surface and intracellular cytokine staining. Surface staining of the cells was performed by incubating with fluorescent conjugated monoclonal antibodies specific to CD3, CD4, and CD8 for 20 min on ice. After surface staining, the cells were washed two times, fixed and permeabilized for 20 min at 4? C. using BD Cytofix/Cytoperm buffer (BD Biosciences) and washed twice with the BD Perm/Wash buffer. Intracellular cytokine staining was then performed by incubating with the fluorescent conjugate antibodies against murine IL-2, IL-5, IL-10, TNF-?, and IFN-? on ice for 30 min. The cells were washed three times and analyzed with a FACS Fortessa flow cytometer (BD Biosciences).

[1019] As shown in FIG. 2A, both mRNA MNR MOMP E FL and mRNA UNR MOMP E DII:IV (2 ?g) were able to induce cross-serovar-responsive CD4+ IFN?+ T cells. Responses for mRNA MNR MOMP E FL (tested at multiple doses) were dose-dependent.

[1020] As shown in FIG. 2B, mRNA MNR MOMP E FL induced cross-serovar-responsive CD8+ IFN?+ cells in a dose-dependent manner. mRNA UNR MOMP E DII:IV (2 ?g dose) elicited minimal CD8+ IFN?+ T cell responses.

[1021] Thus, removal of VD loops may elevate CD4+ IFN?+ T cell responses.

Example 5Immunisation with MOMP mRNA Elicits an Immune Response

[1022] LNP-formulated constructs (all unmodified mRNAs) were used to immunise mice. 8 mice (Balb/c) were immunised in each study group. The following study groups were assessed (human leader=signal sequence of SEQ ID NO: 191): [1023] Group 1: Na?veNegative control (LNP (MC3) buffer) [1024] Group 2: UV EB+CpG prime and live boost (First priming immunisation with Ct elementary bodies (EB) (107 inclusion-forming units (IFU) of UV-inactivated EB)+20 ?g CpG adjuvant, and second boost immunisation with 107 IFU live Ct serovar E EB+20 ?g CpG adjuvant [1025] Group 3: rMOMP (recombinant serovar E MOMP protein) (SEQ ID NO: 134); 50 ?g dose+20 ?g CpG adjuvant [1026] Group 4: MC3 Ser E MOMP (encoding protein of SEQ ID NO: 148); 5 ?g dose [1027] Group 5: MC3 Ser E MOMP (encoding protein of SEQ ID NO: 148); 0.5 ?g dose [1028] Group 6: MC3 Ser E MOMP with human leader (encoding protein of SEQ ID NO: 149); 5 ?g dose [1029] Group 7: MC3 Ser E MOMP with human leader (encoding protein of SEQ ID NO: 149); 0.5 ?g dose [1030] Group 8: OF-02 Ser E MOMP (encoding protein of SEQ ID NO: 151); 5 ?g dose [1031] Group 9: OF-02 Ser E MOMP (encoding protein of SEQ ID NO: 151); 0.5 ?g dose [1032] Group 10: OF-02 Ser E MOMP with human leader (encoding protein of SEQ ID NO: 152); 5 ?g dose [1033] Group 11: OF-02 Ser E MOMP with human leader (encoding protein of SEQ ID NO: 152); 0.5 ?g dose

[1034] Immunisations were given at days 0 and 28, and bleeds were performed at days 0, 14, 28 and 42. Spleens were collected at day 42.

Ability to Elicit IgG that Binds Recombinant MOMP (ELISA)

[1035] Reciprocal dilution IgG titers were assessed in plate ELISA using recombinant serovar E MOMP. The formulation with OF-02 LNP led to higher titers at day 42 than the formulation with MC3 (FIG. 3). Titers for OF-02-formulated constructs at 5 ?g were at levels comparable to those of the EB+CpG and rMOMP+CpG controls.

Ability to Elicit IFN?-Producing T Cells (ELISPOT)

[1036] T cell responses were assessed in splenic IFN? ELISPOT, as shown in FIGS. 4A, B and C. Different recall antigens were used, and are indicated above the individual graphs: UV EB, which stands for UV inactivated EBs (FIG. 4A), rMOMP, which is recombinant serovar E MOMP protein (FIG. 4B), and total peptide pool, which stands for a pool of overlapping serovar E MOMP peptides (as described above) (FIG. 4C).

[1037] These data indicate that immunisation with either OF-02 or MC3 formulated WT serovar E MOMP mRNA elicits MOMP specific IFN?-producing T cells, and these T cells are elicited in response to any of the tested recall antigens.

Example 6MOMP Constructs Lacking One or More VD Loops Induce a T Cell Response

[1038] Further mRNA (UNR) constructs delivering MOMP without variable domains were tested for their ability to elicit T cell responses.

The Removal of the Variable Domains does not Impair the Ability to Elicit an IFN? MOMP T Cell Response

[1039] In this example, mice (female C57BL/6) were immunised with MOMP mRNA constructs (containing unmodified mRNA), or a recombinant MOMP control, to test the effects of the systematic removal of variable domain loops VD1, VD2, VD3, VD4 from MOMP serovar E, either alone or in combination. In the ?VD constructs, nucleic acid sequences were modified so that variable loops were replaced with non-native loop motifs containing glycine residues, in order to remove any immunogenic epitopes contained within the loops. For example, ?VD1 encodes a serovar E MOMP in which the VD1 loop is replaced by six Gly residues. All of the mRNA constructs also encoded the native signal sequence (of SEQ ID NO: 192), with exception of the A-signal-peptide mRNA construct.

[1040] Two studies as set out in the tables below (Table 6 and Table 7) were performed, and the data presented in FIGS. 5A and B, respectively show analysis of splenocytes from mice in these groups:

TABLE-US-00011 TABLE 6 Study groups presented in FIG. 5A Dosing Group Dose LNP or schedule Bleeding schedule N = 10 Antigen (mRNA) (?g) Adjuvant (days) (days) 1 Wild type MOMP 2 OF-02 0, 21 Pre-immunisation - (encoding protein of 1 (Pre-), 21, 34 SEQ ID NO: 154) 2 ?VD4 (encoding 2 OF-02 0, 21 Pre-, 21, 34 protein of SEQ ID NO: 156) 3 ?VD2 ?VD4 2 OF-02 0, 21 Pre-, 21, 34 (encoding protein of SEQ ID NO: 157) 4 ?VD1 ?VD2 ?VD4 2 OF-02 0, 21 Pre-, 21, 34 (encoding protein of SEQ ID NO: 158) 5 ?VD1234 (encoding 2 OF-02 0, 21 Pre-, 21, 34 protein of SEQ ID NO: 159) 6 ?-signal-peptide 2 OF-02 0, 21 Pre-, 21, 34 (encoding protein of SEQ ID NO: 160) Protein Controls 7 rMOMP (SEQ ID 5 AF03 0, 21 Pre-, 21, 34 NO: 134)

TABLE-US-00012 TABLE 7 Study groups presented in FIG. 5B Dosing Bleeding Group Dose LNP or schedule schedule N = 5 Antigen (mRNA) (ug) Adjuvant (days) (days) 1 ?VD1 (encoding protein 2 OF-02 0, 21 Pre-, 21, 35 of SEQ ID NO: 161) 2 ?VD2 (encoding protein 2 OF-02 0, 21 Pre-, 21, 35 of SEQ ID NO: 162) 3 ?VD3 encoding protein 2 OF-02 0, 21 Pre-, 21, 35 of (SEQ ID NO: 163) 4 ?VD2 ?VD3 (encoding 2 OF-02 0, 21 Pre-, 21, 35 protein of SEQ ID NO: 164) 5 ?VD1 ?VD2 ?VD3 2 OF-02 0, 21 Pre-, 21, 35 (encoding protein of SEQ ID NO: 165) 6 ?VD2 ?VD3 ?VD4 2 OF-02 0, 21 Pre-, 21, 35 (encoding protein of SEQ ID NO: 166) 7 ?VD1 ?VD3 ?VD4 2 OF-02 0, 21 Pre-, 21, 35 (encoding protein of SEQ ID NO: 167)

[1041] The ELISPOT assay was used to evaluate the T cell response of the mice after intramuscular (IM) injection, by measuring the level of IFN? production by the T cells collected from the spleens. Spleens from individual mice were collected on the day of the final bleed (34/35) from the mice and T cell responses in splenocytes were assessed by ELISPOT, with pools of overlapping peptides of serovar E MOMP (as described above) used as recall antigen to stimulate the T cell response.

[1042] FIGS. 5A and B show the level of IFN? production by the T cells following immunisation with mLRNA constructs and stimulation with recall antigen. The data indicate that removal of the signal sequence eliminates the T cell response.

[1043] Immunisation with mLRNA encoding MOMP serovar E with one or more of the variable domain loops VD1, VD2, VD3 and VD4 removed, and various combinations thereof, gives rise to a similar, if not enhanced, T cell response to that of FL MOMP serovar E. Therefore, removal of the variable domains does not impair ability to elicit an IFN? MOMP T cell response.

MOMP without Variable Loops VD2 and VD4 is Able to Elicit an Immune Response

[1044] Unmodified (UNR) mRNA encoding MOMP without DII (VD2) and DIV (VD4) domain (DIIDIV), mRNA UNR MOMP E DII:IV (encoding protein of SEQ ID NO: 157), was compared to recombinant MOMP protein (SEQ ID NO: 134) for its ability to induce a T cell response. In the mRNA UNR MOMP E DII:IV construct, variable loops VD2 and VD4 were replaced with non-native loop motifs containing glycine residues, in order to remove any immunogenic epitopes contained within the loops.

[1045] Mice (N=15 in each group) received two immunisations of unmodified (UNR) MOMP deleted of DII and DIV domain (DIIDIV) mRNA native SS-constructs (native sequence signal) (mRNA UNR MOMP E DII:IV) and recombinant MOMP protein (serE) at 2 and 5 ?g dose respectively, formulated with the LNP OF-02 or with a liposome-based adjuvant containing TLR4 agonist (SPA14, as described in WO2022090359) respectively, given by IM route, at 0 and 3 weeks (W0 and W3). A control group (N=15) was injected with PBS. Studies included a negative PBS control. Splenocytes were collected seven, eleven- and fourteen-days post-boost. T specific cellular response was assessed by ELISPOT/FluoroSpot and by ICCS.

Ability to Elicit IFN?-Producing T Cells (FluoroSpot)

[1046] First, ability to elicit IFN?-producing T cells was tested using FluoroSpot. For the FluoroSpot assay, spleen cells (0.2, 0.5 and 1?10.sup.6 per well) were plated in 200 ?l RPMI complete together with and without MOMP peptide pools (as described above) from serovar E in presence of IL2 (Roche) in triplicate cultures each. Subsequently plates were incubated at 37? C., 5% CO.sub.2 for 20 h. After washing with PBS, plates were incubated with detection Ab anti-IFN? (mAb R4-6A2-BAM) for 2h at RT. Image analysis of FluoroSpot assays was performed on Microvision Reader.

[1047] As shown in FIG. 6A, MOMP DIIDIV UNR mRNA (i.e. mRNA UNR MOMP E DII:IV) was able to induce a comparable level of IFN?-producing T cells relative to recombinant MOMP protein as formulated with adjuvant. High levels of IFN?-producing T cells were observed between these antigens at days 7, 11 and 14. Therefore, removal of the variable domains VD2 and VD4 does not impair ability to elicit an IFN? MOMP T cell response relative to rMOMP formulated with SPA14 adjuvant.

Ability to Elicit CD4+ IFN?+ and CD8+ IFN?+ Cell Responses (ICCS)

[1048] The mRNA UNR MOMP E DII:IV construct was also compared to recombinant MOMP protein for its ability to elicit CD4+ IFN?+ cells and CD8+ IFN?+ cells, using ICCS.

[1049] For ICCS, isolated lymphocytes from the D7, D11 and D14 splenocyte samples were activated with specific serovar E-MOMP peptide pool (as described above) at 1?10.sup.6 cells/well in 96-well plates and cultured for 6 h in 5% CO.sub.2 at 37? C. with BFA in order to block cytokine secretion. Activated cells were harvested for surface and intracellular cytokine staining. Surface staining of the cells was performed by incubating with fluorescent conjugated monoclonal antibodies specific to CD3, CD4, and CD8 for 20 min on ice. After surface staining, the cells were washed two times, fixed and permeabilized for 20 min at +4? C. using BD Cytofix/Cytoperm buffer (BD Biosciences) and washed twice with the BD Perm/Wash buffer. Intracellular cytokine staining was then performed by incubating with the fluorescent conjugate antibodies against murine IL-2, IL-5, IL-10, TNF-?, and IFN? on ice for 30 min. The cells were washed three times and analyzed with a FACS Fortessa flow cytometer (BD Biosciences).

[1050] As shown in FIG. 6B, across each of the splenocyte samples (collected 7, 11 and 14 days post-boost, respectively), both MOMP DIIDIV UNR mRNA (i.e. mRNA UNR MOMP E DII:IV) and recombinant MOMP protein were able to elicit the production of CD4+ IFN? cells. As shown in FIG. 6C, MOMP DIIDIV UNR mRNA also elicited a CD8+ IFN? cell response, whereas recombinant MOMP protein did not.

[1051] In conclusion, the MOMP DIIDIV UNR mRNA, like rMOMP, elicits a significant CD4+ IFN? cell response. Additionally, it shows a CD8+ IFN? cell response which is not present for rMOMP.

Example 7T Cell Constructs Elicit a Strong T Cell Response

T Cell Constructs are Able to Elicit a T Cell Response

[1052] 7.1 The following mRNA constructs of MOMP variants (including those designed in Example 1; all modified) were formulated in LNP OF-02 and tested for their ability to produce a T cell response: [1053] MOMP_P3_ssHA1 (P3) (encoding protein of SEQ ID NO: 53) [1054] MOMP_P5_ssHA1_C2S (P5 C2S) (encoding protein of SEQ ID NO: 59) [1055] MOMP_serE_FL_ssHA1 (FL) (encoding protein of SEQ ID NO: 29) [1056] MOMP_serE_FL_ssHA1_C2S_Glycneg (FL C2S Glycneg) (encoding protein of SEQ ID NO: 35) [1057] Empty LNP OF-02

[1058] Mice received two immunisations of mRNA ssHA1-constructs (comprising sequence signal from hemagglutinin H1N1 A/Caledonia/20/1999; ssHA1 is given by SEQ ID NO: 187) at 0.125 ?g dose, formulated with the LNP OF-02, given by IM route, at 0 and 3 weeks (W0 and W3). Studies included a negative empty-LNP control and a positive full-length-MOMP-Serovar E control. Splenocytes were collected two weeks post-boost. A T specific cellular response was assessed by ELISPOT/FluoroSpot and by ICCS.

[1059] 7.2 In a second study, the following mRNA constructs of MOMP variants (including those designed in Example 1; all modified) were formulated in LNP OF-02 and tested for their ability to produce a T cell response: [1060] MOMP_P3_ssHA1 (P3) (encoding protein of SEQ ID NO: 53) [1061] MOMP_P3_ssHA1_C2S (P3 C2S) (encoding protein of SEQ ID NO: 55) [1062] MOMP_P5_ssHA1 (P5) (encoding protein of SEQ ID NO: 57) [1063] MOMP_P5_ssHA1_C2S (P5 C2S) (encoding protein of SEQ ID NO: 59) [1064] MOMP_serE_FL_ssHA1 (Ser E FL) (encoding protein of SEQ ID NO: 29) [1065] MOMP_serD_FL_ssHA1 (Ser D FL) (encoding protein of SEQ ID NO: 21) [1066] Empty LNP OF-02

[1067] Mice received two immunisations of mRNA ssHA1-constructs (comprising sequence signal from hemagglutinin H1N1 A/Caledonia/20/1999; ssHA1 is given by SEQ ID NO: 187) at 0.125 ?g or 2 ?g dose, formulated with the LNP OF-02, given by IM route, at 0 and 3 weeks (W0 and W3). Studies included a negative empty-LNP control and positive full-length-MOMP-Serovar E and full-length-MOMP-Serovar D controls. Splenocytes were collected one week post-boost. A T specific cellular response was assessed by ELISPOT/FluoroSpot (0.125 ?g dose) and by ICCS (2 ?g dose). Results provided in this Example were obtained from the study described in point 7.1, unless indicated otherwise.

Ability to Elicit IFN?-Producing T Cells and IL-5 Response (FluoroSpot)

[1068] First, ability to elicit IFN?-producing and IL-5 producing T cells was tested using FluoroSpot. For the FluoroSpot assay, spleen cells (2?10.sup.5 per well) were plated in 200 ?l RPMI complete together with and without MOMP peptide pools (as described above) from serovar D, E, F, G and J (left to right/light grey to dark grey, respectively in FIGS. 7-8) in the presence of IL2 (Roche) and in triplicate cultures. Subsequently, plates were incubated at 37? C., 5% CO.sub.2 for 20 h. After washing with PBS, plates were incubated with detection Ab anti-IFN? (mAb R4-6A2-BAM), or detection Ab anti-IL-5 (mAb TRFK4, biotin) for 2 h at RT. Image analysis of FluoroSpot assays was performed on Microvision Reader.

[1069] As shown in FIG. 7, MOMP_P3_ssHA1 (P3) and MOMP_P5_ssHA1_C2S (P5 C2S), like MOMP_serE_FL_ssHA1 (FL), gave rise to high numbers of cross-serovar-responsive IFN?-producing T cells. In contrast, MOMP_serE_FL_ssHA1_C2S_Glycneg (FL C2S Glycneg) did not elicit a level of cross-serovar-responsive IFN?-producing T cell response above that of empty LNPs.

[1070] As shown in FIG. 8, all of the immunisation samples (MOMP_P3_ssHA1 (P3), MOMP_P5_ssHA1_C2S (P5 C2S), MOMP_serE_FL_ssHA1 (FL), MOMP_serE_FL_ssHA1_C2S_Glycneg (FL C2S Glycneg) and empty LNP) induced low levels of cross-serovar-responsive IL-5-producing cells, indicating a minimal Th2 response.

[1071] mRNA constructs evaluated in the study detailed in section 7.2 were also tested for their ability to elicit IFN?-producing T cells using the FluoroSpot assay described above with plating of 1?10.sup.5 spleen cells per well.

[1072] As shown in FIG. 9, all mRNA constructs tested in the study detailed in section 7.2 gave rise to high numbers of cross-serovar-responsive IFN?-producing T cells.

Ability to Elicit CD4+ IFN?+, CD8+ IFN?+, and Polyfunctional Cell Responses (ICCS)

[1073] The MOMP mRNA constructs were also tested for their ability to elicit CD4+ IFN?+ cells, CD8+ IFN?+ cells and polyfunctional cells in mice.

[1074] For ICCS, isolated lymphocytes were activated with specific serovar E-MOMP peptide pool (as described above) at 1?10.sup.6 cells/well in 96-well plates and cultured for 6 h in 5% CO.sub.2 at 37? C. with BFA in order to block cytokine secretion. Activated cells were harvested for surface and intracellular cytokine staining. Surface staining of the cells was performed by incubating with fluorescent conjugated monoclonal antibodies specific to CD3, CD4, and CD8 for 20 min on ice. After surface staining, the cells were washed two times, fixed and permeabilized for 20 min at +4? C. using BD Cytofix/Cytoperm buffer (BD Biosciences) and washed twice with the BD Perm/Wash buffer. Intracellular cytokine staining was then performed by incubating with the fluorescent conjugate antibodies against murine IL-2, IL-5 or IL-17, IL-10, TNF-?, and IFN? on ice for 30 min. The cells were washed three times and analyzed with a FACS Fortessa flow cytometer (BD Biosciences).

[1075] As shown in FIG. 10A, MOMP_P3_ssHA1 (P3) and MOMP_P5_ssHA1_C2S (P5 C2S), like MOMP_serE_FL_ssHA1 (FL), gave rise to significant CD4+ IFN? T cell responses. In contrast, MOMP_serE_FL_ssHA1_C2S_Glycneg (FL C2S Glycneg) did not give rise to a CD4+ IFN? T cell response that was distinguishable from the empty LNP control.

[1076] As shown in FIG. 10B, CD8+ IFN? T cell responses were absent or much lower for the tested constructs.

[1077] The MOMP mRNA constructs were also assessed for their ability to induce polyfunctional cells. FIG. 12 shows, for each of the mRNA constructs, the percentage of MOMP-specific CD4+ T cells that were elicited and indicates the relative percentages of CD4+ T cells producing each of IFN? (IFNg), IL2, and/or TNF? (singularly and in the various combinations thereof). For each of MOMP_P3_ssHA1 (P3), MOMP_P5_ssHA1_C2S (P5 C2S) and MOMP_serE_FL_ssHA1 (FL), the CD4+ IFN?+IL2+TNF?+ cell population formed the majority, relative to the other cell populations. Little to no IL5 or IL10 was observed for any of the tested constructs (data not shown).

[1078] The MOMP mRNA constructs evaluated in the study described in section 7.2, were similarly tested for their ability to elicit MOMP-specific CD4+ IFN?+ cells, CD8+ IFN?+ T cells and polyfunctional cells in mice.

[1079] As shown in FIG. 11A, all MOMP mRNA constructs gave rise to a significant CD4+ IFN? T cell response that was distinguishable from the empty LNP control. The CD4+ IFN? T cell response was slightly higher for MOMP_P3_ssHA1 (P3), MOMP_P5_ssHA1 (P5), MOMP_P3_ssHA1_C2S (P3 C2S), and MOMP_P5_ssHA1_C2S (P5 C2S) as compared to the full length MOMP mRNA constructs.

[1080] As shown in FIG. 11B, CD8+ IFN? T cell responses were absent or much lower for the tested constructs, with the exception of the MOMP_P5_ssHA1_C2S (P5 C2S) and MOMP_serD_FL_ssHA1 (SerD FL) constructs.

[1081] The MOMP mRNA constructs evaluated in the study described in section 7.2, were also assessed for their ability to induce polyfunctional cells. FIG. 13 shows, for each of the MOMP mRNA constructs, the percentage of MOMP-specific CD4+ T cells that were elicited and indicates the relative percentages of CD4+ T cells producing each of IFN? (IFNg), IL2, and/or TNF? (singularly and in the various combinations thereof). For all of the MOMP mRNA constructs tested, the CD4+ IFN?+IL2+TNF?+ cell population formed the majority, relative to the other cell populations. Little to no IL-17 or IL-10 was observed for any of the tested constructs (data not shown).

T Cell Constructs Elicit Low Levels of rMOMP Binding Antibodies

[1082] The MOMP mRNA constructs were also evaluated to determine if they elicited recombinant MOMP (rMOMP) binding antibodies. Blood samples were collected at 0 weeks (W0) and one week after the last vaccine administration, according to the protocol described in section 7.2 above, in order to measure total IgG by ELISA. The specific IgG were measured from individual sera using automated 384 ELISA.

[1083] Briefly, 384-well micro-plates were coated in carbonate buffer with 20 ?L per well recombinant MOMP protein at 2 ?g/mL and kept overnight at +4? C. Coating solution was removed and washed with buffer 1 (PBS/Tween 0.05%). Free sites were blocked with 75 ?L of buffer 2 (PBS/Tween 20 at 0.05%/milk 1%) and incubated 90 min at room temperature (RT). Plates were emptied, then sera were serially diluted in buffer 2 under a volume of 20 ?L (12 times) in the microplates. The plates were incubated for 90 min at RT and then washed with buffer 1. 20 ?l of a diluted anti-mouse total IgG peroxidase conjugate (Southern Biotech) was added in each well (1/2000). After 90 min incubation at RT, the plates were washed with buffer 1. The reaction was developed by adding 20 ?L of a tetramethylbenzidine substrate solution in each well. The reaction was chemically stopped after 30 mD at RT with HCl (1N (normality)) and absorbance was measured at 450-650 nm on a spectrophotometer (Synergy HTX, Biotek). The results were analysed in Softmax Pro software using a standard curve and expressed in arbitrary ELISA units by the reciprocal dilution corresponding to an OD of 1.

[1084] As shown in FIG. 14, the MOMP SRNA constructs (P3, P3 C2S, P5, and P5 C2S) tested elicited low levels of recombinant MOMP binding antibodies. Levels were similar to those observed in pre-immune sera or with empty LNPs, as expected given that these constructs lacked variable domain loops.

Example 8Immunisation with Non-MOMP Antigens Induces Immune Responses

Non-MOMP Ct Protein Constructs Induce a T Cell Response

[1085] Mice (female C57BL/6) were immunised with various Ct non-MOMP recombinant proteins, alongside controls of recombinant MOMP protein or MOMP ?VD1234 mRNA. The study groups are set out in Table 8 below:

TABLE-US-00013 TABLE 8 Study groups Dosing Bleeding Group Dose schedule schedule N = 5 Antigen (ug) Administration Adjuvant (days) (days) 1 rCT584; SEQ 2 Subcutaneous 10 mg CpG/mouse/ 0, 21 Pre-, 21, 37 ID NO: 145 (SC) injection + IFA 2 rCT812; SEQ 2 SC 10 mg CpG/mouse/ 0, 21 Pre-, 21, 37 ID NO: 197 injection + IFA 3 rCT600; SEQ 2 SC 10 mg CpG/mouse/ 0, 21 Pre-, 21, 37 ID NO: 146 injection + IFA 4 rCT443; SEQ 2 SC 10 mg CpG/mouse/ 0, 21 Pre-, 21, 37 ID NO: 198 injection + IFA 5 Viable EB 1e7* SC 10 mg CpG/mouse/ 0, 21 Pre-, 21, 37 (E/Bour injection + IFA ATCC VR-348B) 6 rMOMP; SEQ 2 SC 10 mg CpG/mouse/ 0, 21 Pre-, 21, 37 ID NO: 134 injection + IFA 7 MOMP ?VD1234 2 Intramuscular OF-02 0, 21 Pre-, 21, 37 (UNR mRNA) (IM) (encoding protein of SEQ ID NO: 159)

[1086] The ELISPOT assay was used to evaluate the T cell response of the mice after immunisation, by measuring the level of IFN? production produced by the T cells collected from the spleens. Spleens from individual mice were collected from the mice and T cell responses in splenocytes were assessed by ELISPOT, with and without pools of overlapping peptides as described above of the relevant specified native MOMP or non-MOMP used as recall antigen to stimulate the T cell response.

[1087] As shown in FIG. 15, recombinant (r) non-MOMP proteins rCT584, rCT812, rCT600 and rCT443 each induce IFN?-producing T cells at much greater levels than EB. Responses elicited by recombinant MOMP protein (rMOMP; SEQ ID NO: 134) and mRNA encoding MOMP serovar E lacking VD loops 1-4 (mRNA MOMP ?VD1234) are included for comparison.

mRNA Constructs Encoding Non-MOMP Ct Antigens Induce a T Cell Response

[1088] Mice (female C57BL/6) were immunised (IM) with unmodified (UNR) mRNA encoding various Ct non-MOMP antigen fragments alongside the mRNA MOMP ?VD1234 construct, formulated in cKK-e10-based LNPs. LNP cKK-e10 (Lipid B)=cationic lipid cKK-e10 at a molar ratio of 40%; DMG-PEG2000 at a molar ratio of 1.5%; cholesterol at a molar ratio of 28.5%; and DOPE at a molar ratio of 30%.

[1089] The study groups are set out in Table 9 below:

TABLE-US-00014 TABLE 9 Study groups Group Antigen Dose Dosing Bleeding N = 5 (all unmodified mRNA) (ug) LNP schedule schedule 1 CT812: 1-1530 (encoding 2 cKK-e10 0, 21 Pre-, 21, 35 protein of SEQ ID NO: 168) 2 CT812: 1-761 (encoding 2 cKK-e10 0, 21 Pre-, 21, 35 protein of SEQ ID NO: 169) 3 Nan96-SS:CT812: 32-761 2 cKK-e10 0, 21 Pre-, 21, 35 (encoding protein of SEQ ID NO: 170) 4 CT600: 1-188 (encoding 2 cKK-e10 0, 21 Pre-, 21, 35 protein of SEQ ID NO: 171) 5 Nan96-SS:CT600: 2-188 2 cKK-e10 0, 21 Pre-, 21, 35 (encoding protein of SEQ ID NO: 172) 6 CT443: 1-576 (encoding 2 cKK-e10 0, 21 Pre-, 21, 35 protein of SEQ ID NO: 173) 7 Nan96-SS:CT443: 32-576 2 cKK-e10 0, 21 Pre-, 21, 35 (encoding protein of SEQ ID NO: 174) 8 MOMP ?VD1234 (encoding 2 cKK-e10 0, 21 Pre-, 21, 35 protein of SEQ ID NO: 159)

[1090] Nan96-SS is the ssHA1 secretion signal given by SEQ ID NO:187. Numbering after the colons indicates the start and end residue (numbering relative to wild-type) of the relevant fragment of each of non-MOMP proteins CT443, CT600, and CT812. In the Nan96-SS constructs, the nucleic acid sequences encoding the ssHA1 secretion signal of SEQ ID NO: 187 and the non-MOMP protein fragment are linked by a nucleic acid sequence encoding the amino acids DTI.

[1091] The ELISPOT assay was used to evaluate the T cell response of the mice after immunisation, by measuring the level of IFN? production produced by the T cells collected from the spleens. Spleens from individual mice were collected from the mice (day 35) and T cell responses in splenocytes were assessed by ELISPOT, with pools of overlapping peptides to the relevant specified MOMP or non-MOMP (as described above) used as recall antigen to stimulate the T cell response.

[1092] As shown in FIG. 16, all of the non-MOMP fragment mRNA constructs (CT812: 1-1530, CT812: 1-761, Nan96-SS:CT812: 32-761, CT600: 1-188, CT443: 1-576, Nan96-SS:CT443: 32-576), gave rise to comparable levels of IFN?-producing T cells to the mRNA encoding MOMP serovar E lacking VD loops 1-4 (MOMP ?VD1234), with exception of Nan96-SS:CT600: 2-188.

[1093] Therefore, various mRNA constructs encoding non-MOMP Ct antigen fragments are also able to elicit a significant IFN?-producing T cell response.

[1094] mRNA Constructs Encoding Non-MOMP Ct Antigens Induce a T Cell Response mRNA encoding non-MOMP Ct antigens CT443 (Nan96-SS:CT443: 32-576), CT584 (Nan96HA-SS:CT584:2-183), CT600 (Nan96HA-SS:CT600:2-188) and CT812 (Nan96-SS:CT812:32-761) were compared to recombinant MOMP protein for their ability to induce a T cell response.

[1095] Mice received two immunisations of unmodified mRNA native signal sequence constructs at 2 ?g dose, formulated with the cKK-e10-based LNP, given by IM route, at 0 and 3 weeks (W0 and W3). Study included a positive full-length-MOMP-Serovar E recombinant protein control (SEQ ID NO: 134) (5 ?g)+SPA14. Splenocytes were collected at days eleven and fourteen post-boost. T specific cellular response was assessed by ELISPOT/FluoroSpot and by ICCS.

Ability to Elicit IFN?-Producing T Cells (FluoroSpot)

[1096] First, ability to elicit IFN?-producing T cells was tested using FluoroSpot. For the FluoroSpot assay, spleen cells (2?10.sup.5 per well) were plated in 200 ?l RPMI complete together with and without respective peptide pools (as described above) in presence of IL2 (Roche) at 0.5 to 2 ?g/mL in triplicate cultures each. Subsequently plates were incubated at 37? C., 5% CO.sub.2 for 20 h. After washing with PBS, plates were incubated with detection Ab anti-IFN? (mAb R4-6A2-BAM) for 2 h at RT. Image analysis of FluoroSpot assays was performed on Microvision Reader.

[1097] As shown in FIG. 17, mRNA encoding non-MOMP Ct antigens CT443, CT584 and CT812 were, like recombinant MOMP serovar E protein, able to elicit high levels of IFN?-producing T cells. Responses to mRNA encoding non-MOMP Ct antigen CT600 were lower.

[1098] Therefore, various mRNA constructs encoding native non-MOMP Ct antigens (particularly CT443, CT584 and CT812) are able to elicit a strong IFN?-producing T cell response.

Ability to Elicit CD4+ IFN?+ and CD8+ IFN?+ Cell Responses (ICCS)

[1099] The same constructs were also tested for their ability to elicit CD4+ IFN?+ cells and CD8+ IFN?+ cells in mice.

[1100] For ICCS, isolated lymphocytes were activated with specific respective peptide pools (as described above) at 1?10.sup.6 cells/well in 96-well plates and cultured for 6 h in 5% CO.sub.2 at 37? C. with BFA in order to block cytokine secretion. Activated cells were harvested for surface and intracellular cytokine staining. Surface staining of the cells was performed by incubating with fluorescent conjugated monoclonal antibodies specific to CD3, CD4, and CD8 for 20 min on ice. After surface staining, the cells were washed two times, fixed and permeabilized for 20 min at +4? C. using BD Cytofix/Cytoperm buffer (BD Biosciences) and washed twice with the BD Perm/Wash buffer. Intracellular cytokine staining was then performed by incubating with the fluorescent conjugate antibodies against murine IL-2, IL-5, IL-10, TNF-?, and IFN? on ice for 30 min. The cells were washed three times and analyzed with a FACS Fortessa flow cytometer (BD Biosciences).

[1101] As shown in FIG. 18A, across each of the splenocyte samples (1 or 2 ug doses and collected at 11 or 14 days post-boost), mRNA encoding non-MOMP antigens CT443, CT584, CT600 and CT812 gave rise to a CD4+ IFN? cell response, but which was lower than that elicited by recombinant MOMP protein.

[1102] As shown in FIG. 18B, across each of the splenocyte samples (1 or 2 ug doses and collected at 11 or 14 days post-boost), mRNA encoding non-MOMP antigens CT584 and CT812 elicited high CD8+ IFN? cell responses relative to CT443, CT600 and recombinant MOMP protein.

Non-MOMP Ct Antigen Constructs are Able to Induce Specific IgG Against their Encoded Proteins

[1103] In this example, the following mRNA constructs encoding non-MOMP Ct antigens (all modified) were tested for their ability to induce specific IgG against their respective protein: CT443_ssHA1 (CT443; encoding protein of SEQ ID NO:105), CT443_ssHA1_GlycNeg (CT443_GN, encoding protein of SEQ ID NO:106), CT584_ssHA1 (CT584, encoding protein of SEQ ID NO:107), CT584_ssHA1_Glycneg (CT584_GN, encoding protein of SEQ ID NO:108), CT584_ssHA1_C2S (CT584_C2S, encoding protein of SEQ ID NO:109), CT584_ssHA1_Glycneg_C2S (CT584_GN_C2S, encoding protein of SEQ ID NO:110), CT600_trunc_ssHA1 (CT600_trunc; encoding protein of SEQ ID NO: 111), CT600_trunc_ssHA1_Glycneg (CT600_trunc_GN, encoding protein of SEQ ID NO:112), CT812_pass-domain_ssHA1 (CT812_pass-dom, encoding protein of SEQ ID NO: 113), CT812_pass-domain_ssHA1_C2S (CT812_pass-dom_C2S, encoding protein of SEQ ID NO: 114), CT812_ext-pass-domain_ssHA1 (CT812_ext-pass-dom, encoding protein of SEQ ID NO: 115), CT812_ext-pass-domain_ssHA1_C2S (CT812_ext-pass-dom_C2S, encoding protein of SEQ ID NO: 116).

[1104] Mice (C57Bl/6) received two immunisations of mRNA constructs at 5 ?g dose, formulated with the LNP OF-02, given by IM route, at 0 and 3 weeks (W0 and W3) in at least one of two independent studies. Studies included a negative empty OF-02 LNP control. Blood samples were collected at 0 weeks (W0) and two weeks after the last vaccine administration, in order to measure total IgG by ELISA. The specific IgG were measured from individual sera using automated 384 ELISA.

[1105] Briefly, 384-well micro-plates were coated in carbonate buffer with 20 ?L per well of respective protein (CT443 (4 ?g/mL), CT584 (1 ?g/mL), CT600 (4 ?g/mL)) and kept overnight at +4? C. Coating solution was removed and washed with buffer 1 (PBS/Tween 0.05%). Free sites were blocked with 75 ?L of buffer 2 (PBS/Tween 20 at 0.05%/milk 1%) and incubated 90 min at room temperature (RT). Plates were emptied, then sera were serially diluted in buffer 2 under a volume of 20 ?L (12 times) in the microplates. The plates were incubated for 90 min at RT and then washed with buffer 1. 20 ?l of a diluted anti-mouse total IgG peroxidase conjugate (Southern Biotech) was added in each well (1/1000). After 90 min incubation at RT, the plates were washed with buffer 1. The reaction was developed by adding 20 ?L of a tetramethylbenzidine substrate solution in each well. The reaction was chemically stopped after 30 min at RT with HCl (1N (normality)) and absorbance was measured at 450-650 nm on a spectrophotometer (Synergy HTX, Biotek). The results were analyzed in Softmax Pro software using a standard curve and expressed in arbitrary ELISA units by the reciprocal dilution corresponding to an OD of 1.

[1106] As shown in FIG. 19, all samples tested gave rise to high titers of IgGs which were specific against their respective protein, with the exception of CT812, which elicited lower IgG titers for all constructs.

mRNA Constructs Encoding Non-MOMP Ct Antigens Induce a T Cell Response

[1107] Further mRNA constructs (all modified) encoding non-MOMP Ct antigens (CT443_ssHA1 (CT443, encoding protein of SEQ ID NO: 105), CT584_ssHA1_GlycNeg (CT584_GlycNeg, encoding protein of SEQ ID NO:108), CT600_trunc_ssHA1_Glycneg (CT600_GlycNeg, encoding protein of SEQ ID NO:112) were tested for their ability to induce a T cell response, as compared to a control of mRNA encoding MOMP_serE_FL_ssHA1 (MOMP ser E FL; encoding protein of SEQ ID NO: 29).

[1108] Mice received two immunisations of mRNA ssHA1-constructs at 0.125 ?g dose, formulated with the LNP OF-02, given by IM route, at 0 and 3 weeks (W0 and W3). Study included a negative empty-LNP (OF-02) and a positive MOMP_serE_FL_ssHA1 control. Splenocytes were collected two weeks post-boost and T specific cellular response was assessed by ELISPOT/FluoroSpot and by ICCS.

Ability to Elicit IFN?-Producing T Cells (FluoroSpot)

[1109] First, ability to elicit IFN?-producing T cells was tested using FluoroSpot. For the FluoroSpot assay, spleen cells (2?10.sup.5 per well) were plated in 200 ?l RPMI complete together with and without respective native peptide pools (as described above) in presence of IL2 (Roche) in triplicate cultures each. Subsequently plates were incubated at 37? C., 5% CO.sub.2 for 20 h. After washing with PBS, plates were incubated with detection Ab anti-IFN? (mAb R4-6A2-BAM) for 2 h at RT. Image analysis of FluoroSpot assays was performed on Microvision Reader.

[1110] As shown in FIG. 20, the CT443 and CT584_GlycNeg constructs were able to induce similarly high, if not enhanced, levels of IFN?-producing T cells, relative to the MOMP serE FL construct. However, CT600_GlycNeg did not induce similarly high levels of IFN?-producing T cells.

Ability to Elicit CD4+ IFN?+, CD8+ IFN?+, and Polyfunctional Cell Responses (ICCS)

[1111] For ICCS, isolated lymphocytes were activated with specific respective peptide pools (as described above) at 1?10.sup.6 cells/well in 96-well plates and cultured for 6 h in 5% CO.sub.2 at 37? C. with BFA in order to block cytokine secretion. Activated cells were harvested for surface and intracellular cytokine staining. Surface staining of the cells was performed by incubating with fluorescent conjugated monoclonal antibodies specific to CD3, CD4, and CD8 for 20 min on ice. After surface staining, the cells were washed two times, fixed and permeabilized for 20 min at +4? C. using BD Cytofix/Cytoperm buffer (BD Biosciences) and washed twice with the BD Perm/Wash buffer. Intracellular cytokine staining was then performed by incubating with the fluorescent conjugate antibodies against murine IL-2, IL-5, IL-10, TNF-?, and IFN-? on ice for 30 min. The cells were washed three times and analyzed with a FACS Fortessa flow cytometer (BD Biosciences).

[1112] As shown in FIG. 21A, the CT443 construct gave rise to a CD4+ IFN? T cell response, albeit at a lower level relative to MOMP_serE_FL_ssHA1 (MOMP ser E FL). Immunisation with either of CT584_GlycNeg or CT600_GlycNeg did not appear to give rise to a CD4+ IFN? T cell response.

[1113] As shown in FIG. 21B, the CT443 and CT584_GlycNeg constructs each gave rise to a relatively strong CD8+ IFN? T cell response. MOMP_serE_FL_ssHA1 (MOMP ser E FL) and CT600_GlycNeg CD8+ IFN? T cell responses were indistinguishable from empty LNP control.

[1114] The mRNA constructs were also assessed for their ability to induce polyfunctional cells. FIG. 22 shows, for each of the mRNA constructs, the percentages of CD4+(A) and CD8+(B) antigen-specific T cells that were elicited and indicates the percentage of CD4+ and CD8+ T cells producing each of IFN? (IFNg), IL2, and/or TNF? (TNF?) (singularly and in the various combinations thereof). CD4+ responses were elicited for each of MOMP_serE_FL_ssHA1 (MOMP ser E FL) and CT443, and the polyfunctional CD4+ IFN?+IL2+TNF?+ cell population formed the majority, relative to the other CD4+ T cell populations (FIG. 22A).

[1115] CD8+ responses were elicited for each of CT443 and CT584_GlycNeg, and the CD8+ IFN?+IL2-TNF?+ cell population formed the majority, relative to the other CD8+ T cell populations (FIG. 22B).

Non-MOMP Ct Antigen Constructs are Able to Induce Specific IgG Against their Encoded Proteins at a Low Dose of 0.125 ?g

[1116] Further non-MOMP Ct antigens were tested for their ability to induce specific IgG against their respective protein. mRNA constructs (all modified) included: CT443_ssHA1 (CT443, encoding protein of SEQ ID NO: 105, CT584_ssHA1_GlycNeg (CT584 mRNA, encoding protein of SEQ ID NO: 108), CT600_trunc_ssHA1_Glycneg (CT600 mRNA, encoding protein of SEQ ID NO:112). Protein constructs included: Recombinant (rProt) CT584 (SEQ ID NO: 145), rProt CT600 (SEQ ID NO: 146) and rProt CT443 (SEQ ID NO: 143).

[1117] Mice received two immunisations of mRNA constructs and recombinant protein at 0.125 and 5 ?g dose respectively, formulated with the LNP OF-02 and SPA14 respectively, given by IM route, at 0 and 3 weeks (W0 and W3). Studies included a negative empty OF-02 LNP control. Blood samples were collected at 0 weeks (W0) and two weeks after the last vaccine administration, in order to measure total IgG by ELISA. The specific IgG were measured from individual sera using automated 384 ELISA.

[1118] Briefly, 384-well micro-plates were coated in carbonate buffer with 20 ?L per well of respective protein (CT443 (4 ?g/mL), CT584 (1 ?g/mL), CT600 (4 ?g/mL)) and kept overnight at +4? C. Coating solution was removed and washed with buffer 1 (PBS/Tween 0.05%). Free sites were blocked with 75 ?L of buffer 2 (PBS/Tween 20 0.05%/milk 1%) and incubated 90 min at room temperature (RT). Plates were emptied, then sera were serially diluted in buffer 2 under a volume of 20 ?L (12 times) in the microplates. The plates were incubated for 90 min at RT and then washed with buffer 1. 20 ?l of a diluted anti-mouse total IgG peroxidase conjugate (Southern Biotech) was added in each well (1/1000). After 90 min incubation at RT, the plates were washed with buffer 1. The reaction was developed by adding 20 ?L of a tetramethylbenzidine substrate solution in each well. The reaction was chemically stopped after 30 min at RT with HCl (1N (normality)) and absorbance was measured at 450-650 nm on a spectrophotometer (Synergy HTX, Biotek). The results were analyzed in Softmax Pro software using a standard curve and expressed in arbitrary ELISA units by the reciprocal dilution corresponding to an OD of 1.

[1119] As shown in FIG. 23, all samples tested gave rise to high titers of IgGs which were specific against their respective protein, even at the low dose of 0.125 ?g.

Non-MOMP Ct Antigen Constructs Elicit Antibodies Binding to Elementary Bodies (EBs)

[1120] The following mRNA constructs of non-MOMP Ct antigens (all modified mRNA) were tested for their ability to induce IgGs binding EBs of serovars E, D, or G. Constructs were formulated with the LNP OF-02. 8 or 10 mice (C57BL/6) were used in each study group and 5 mice were used for each negative control.

[1121] The following constructs were tested: [1122] CT443_ssHA1 (CT443, encoding protein of SEQ ID NO:105), [1123] CT443_ssHA1_GlycNeg (CT443_GN, encoding protein of SEQ ID NO:106), [1124] CT584_ssHA1 (CT584, encoding protein of SEQ ID NO:107), [1125] CT584_ssHA1_Glycneg (CT584_GN, encoding protein of SEQ ID NO:108), [1126] CT584_ssHA1_C2S (CT584_C2S, encoding protein of SEQ ID NO:109), [1127] CT600_trunc_ssHA1 (CT600_trunc, encoding protein of SEQ ID NO: 111), [1128] CT600_trunc_ssHA1_Glycneg (CT600_trunc_GN, encoding protein of SEQ ID NO:112) in two independent studies, [1129] CT812_pass-domain_ssHA1 (CT812_pass-dom, encoding protein of SEQ ID NO: 113), [1130] CT812_pass-domain_ssHA1_C2S (CT812_pass-dom_CS2, encoding protein of SEQ ID NO: 114), [1131] CT812_ext-pass-domain_ssHA1 (CT812_ext-pass-dom, encoding protein of SEQ ID NO: 115) in two independent studies, [1132] CT812_ext-pass-domain_ssHA1_C2S (CT812_ext-pass-dom_C2S, encoding protein of SEQ ID NO: 116).

[1133] Mice received two immunisations of mRNA constructs at 5 ?g dose, formulated with the LNP OF-02, given by IM route, at 0 and 3 weeks (W0 and W3) in at least one of two independent studies. Blood samples were collected at 0 weeks (W0) and two weeks after the last vaccine administration, in order to measure total IgG by ELISA. The specific IgG were measured from individual sera using automated 384 ELISA.

[1134] Briefly, 384-well micro-plates were coated in carbonate buffer with 20 ?L per well of EBs at 5-15 ?g/mL depending on the serovar (5 ?g/mL for EBs of serovar G, 7,5 ?g/mL for EBs of serovar D, and 15 ?g/mL for EBs of serovar E) and kept overnight at +4? C. Coating solution was removed and washed with buffer 1 (PBS/Tween 0.05%). Free sites were blocked with 75 ?L of buffer 2 (PBS/Tween 20 0.05%/milk 1%) and incubated 90 min at room temperature (RT). Plates were emptied, then sera were serially diluted in buffer 2 under a volume of 20 ?L (12 times) in the microplates. The plates were incubated for 90 min at RT and then washed with buffer 1. 20 ?L of a diluted anti-mouse total IgG peroxidase conjugate (Jackson Inc) was added in each well (1/2500). After 90 min incubation at RT, the plates were washed with buffer 1. The reaction was developed by adding 20 ?L of a tetramethylbenzidine substrate solution in each well. The reaction was chemically stopped after 30 min at RT with HCl (1N (normality)) and absorbance was measured at 450-650 nm on a spectrophotometer (Synergy HTX, Biotek). The results were analyzed in Softmax Pro software using a standard curve and expressed in arbitrary ELISA units by the reciprocal dilution corresponding to an OD of 1.

[1135] As shown in FIG. 24, various non-MOMP Ct mRNA constructs elicited IgGs which bound to EBs of serovar E. In particular, the CT443, CT584, and CT600 constructs induced acceptable levels of antibodies against EBs of serovar E, which were higher than background levels. Of the CT812 constructs, only the CT812_pass-dom construct induced an acceptable level of antibodies against EBs of serovar E; however, the level remained lower than those observed for the CT443, CT584, and CT600 constructs.

[1136] As shown in FIGS. 25 and 26, CT443 and CT584 constructs also induced acceptable levels of antibodies which bound to EBs of serovars D and G.

Example 9B Cell Constructs are Able to Induce Specific IgG Against MOMP Ser E

[1137] B cell constructs (all modified mRNA) were tested for their ability to induce specific IgG against recombinant MOMP protein (SEQ ID NO: 134).

[1138] The following constructs were tested: [1139] 1. Hirep2_ssHA1 (encoding protein of SEQ ID NO: 93) [1140] 2. MOMP_VDcomb1-extP_ssHA1 (MOMP_C1_P_SSHA1, encoding protein of SEQ ID NO: 61) [1141] 3. MOMP_VDcomb1-extS_ssHA1 (MOMP_C1_S_SSHA1, encoding protein of SEQ ID NO: 69) [1142] 4. MOMP_VDcomb1-extP_ssHA1_TMB1_Glycneg (MOMP_C1_P_GN_TMB1_SSHA1; encoding protein of SEQ ID NO: 67) [1143] 5. MOMP_VDcomb1-extS_ssHA1_TMB1_Glycneg (MOMP_C1_S_GN_TMB1_SSHA1; encoding protein of SEQ ID NO: 75) [1144] 6. MOMP_VDcomb2-extP_ssHA2_TMB2_Glycneg (MOMP_C2_P_GN_TMB2_SSHA2; SEQ ID NO: 84) [1145] 7. MOMP_VDcomb2-extP_ssHA1_Glycneg (MOMP_C2_P_GN_SSHA1, encoding protein of SEQ ID NO: 79) [1146] 8. MOMP_VDcomb2-extP_ssHA2_Glycneg (MOMP_C2_P_GN_SSHA2, encoding protein of SEQ ID NO: 80) [1147] 9. MOMP_VDcomb2-extS_ssHA1_Glycneg (MOMP_C2_S_GN_SSHA1, encoding protein of SEQ ID NO: 87) [1148] 10. MOMP_VDcomb2-extP_ssHA1_TMB1_Glycneg (MOMP_C2_P_GN_TMB1_SSHA1, encoding protein of SEQ ID NO: 83) [1149] 11. MOMP_VDcomb2-extS_ssHA1_TMB1 (MOMP_C2_S_TMB1_SSHA1, encoding protein of SEQ ID NO: 89) [1150] 12. MOMP_serD_FL_ssHA1_C2S_Glycneg (MOMP_serD_FL_C2S_GN_SSHA1, encoding protein of SEQ ID NO: 27) [1151] 13. MOMP_serG_FL_ssHA1_Glycneg (MOMP_serG_FL_GN_SSHA1, encoding protein of SEQ ID NO: 49) [1152] 14. MOMP_serE_FL_ssHA1 (encoding protein of SEQ ID NO: 29) [1153] 15. MOMP_serE_FL_ssHA1_C2S_Glycneg (MOMP_serE_FL_C2S_GN_SSHA1, encoding protein of SEQ ID NO: 35) [1154] 16. recombinant MOMP protein (rMOMP; SEQ ID NO: 134) [1155] 17. empty LNP

[1156] Mice received two immunisations of mRNA constructs at 5 ?g dose, formulated with the LNP OF-02, given by IM route, at 0 and 3 weeks (W0 and W3). Studies included a negative empty-LNP control. Blood samples were collected at 0 weeks (W0) and two weeks after the last vaccine administration, in order to measure total IgG by ELISA. The specific IgG were measured from individual sera using automated 384 ELISA.

[1157] Briefly, 384-well micro-plates were coated in carbonate buffer with 20 ?L per well of recombinant MOMP protein (rMOMP; SEQ ID NO: 134) at 2 ?g/mL and kept overnight at +4? C. Coating solution was removed and washed with buffer 1 (PBS/Tween 0.05%). Free sites were blocked with 75 ?L of buffer 2 (PBS/Tween 20 0.05%/milk 1%) and incubated 90 min at room temperature (RT). Plates were emptied, then sera were serially diluted in buffer 2 under a volume of 20 ?L (12 times) in the microplates. The plates were incubated for 90 min at RT and then washed with buffer 1. 20 ?L of a diluted anti-mouse total IgG peroxidase conjugate (Southern Biotech) was added in each well (1/1000). After 90 min incubation at RT, the plates were washed with buffer 1. The reaction was developed by adding 20 ?L of a tetramethylbenzidine substrate solution in each well. The reaction was chemically stopped after 30 min at RT with HCl (1N (normality)) and absorbance was measured at 450-650 nm on a spectrophotometer (Synergy HTX, Biotek). The results were analyzed in Softmax Pro software using a standard curve and expressed in arbitrary ELISA units by the reciprocal dilution corresponding to an OD of 1.

[1158] As shown in FIG. 27, all samples tested gave rise to high titers of IgGs which were specific against recombinant MOMP, with exception of the MOMP_SerG_FL_ssHA1 construct, which did not give rise to an anti-MOMP IgG response.

Example 10B Cell Constructs Elicit Antibodies Binding to Elementary Bodies (EBs)

[1159] B cell constructs (all modified mRNA) were tested for their ability to induce IgGs binding to EBs of serovars E, D, or G. Constructs were formulated with the LNP OF-02, while the recombinant MOMP protein (positive control) was formulated with a liposome-based adjuvant containing TLR4 agonist (SPA14, as described in WO2022090359).

[1160] The following constructs were tested: [1161] 1. MOMP_VDcomb1-extP_ssHA1 (P of Chimera 1, encoding protein of SEQ ID NO: 61) [1162] 2. MOMP_VDcomb1-extS_ssHA1 (S of Chimera 1, encoding protein of SEQ ID NO: 69) [1163] 3. MOMP_VDcomb1-extP_ssHA1_Glycneg (P_GN of Chimera 1; encoding protein of SEQ ID NO: 63) [1164] 4. MOMP_VDcomb1-extS_ssHA1_Glycneg (S_GN of Chimera 1; encoding protein of SEQ ID NO: 71) [1165] 5. MOMP_VDcomb1-extP_ssHA1_TMB1_Glycneg (P_GN_TMB1 of Chimera 1; encoding protein of SEQ ID NO: 67) [1166] 6. MOMP_VDcomb1-extS_ssHA1_TMB1_Glycneg (S_GN_TMB1 of Chimera 1; encoding protein of SEQ ID NO: 75) [1167] 7. MOMP_VDcomb2-extP_ssHA1 Glycneg (P_GN of Chimera 2, encoding protein of SEQ ID NO: 79) [1168] 8. MOMP_VDcomb2-extS_ssHA1_Glycneg (S_GN of Chimera 2, encoding protein of SEQ ID NO: 87) [1169] 9. MOMP_VDcomb2-extP_ssHA1_TMB1_Glycneg (P_GN_TMB1 of Chimera 2, encoding protein of SEQ ID NO: 83) [1170] 10. MOMP_VDcomb2-extS_ssHA1_TMB1 (S_TMB1 of Chimera 2, encoding protein of SEQ ID NO: 89) [1171] 11. MOMP_serD_FL_ssHA1 (Ser_D, encoding protein of SEQ ID NO: 21) [1172] 12. MOMP_serE_FL_ssHA1 (Ser_E, encoding protein of SEQ ID NO: 29) [1173] 13. MOMP_serF_FL_ssHA1 (Ser_F, encoding protein of SEQ ID NO: 37) [1174] 14. MOMP_serG_FL_ssHA1 (Ser_G, encoding protein of SEQ ID NO: 45)

[1175] Mice (C57BL/6) received two immunisations of mRNA constructs at 5 ?g dose, formulated with the LNP OF-02, given by IM route, at 0 and 3 weeks (W0 and W3) in at least one of two independent studies. Studies included a negative empty-LNP control and a positive full-length recombinant MOMP protein control (rMOMP; SEQ ID NO: 134, indicated as rec Ser E in FIGS. 28-30). Blood samples were collected at 0 weeks (W0) and two weeks after the last vaccine administration, in order to measure total IgG by ELISA. The specific IgG were measured from individual sera using automated 384 ELISA.

[1176] Briefly, 384-well micro-plates were coated in carbonate buffer with 20 ?L per well of EBs at 5-15 ?g/mL per serovar (5 ?g for EBs of serovar G, 7,5 ?g for EBs of serovar D, and 15 ?g for EBs of serovar E) and kept overnight at +4? C. Coating solution was removed and washed with buffer 1 (PBS/Tween 0.05%). Free sites were blocked with 75 ?L of buffer 2 (PBS/Tween 20 0.05%/milk 1%) and incubated 90 min at room temperature (RT). Plates were emptied, then sera were serially diluted in buffer 2 under a volume of L (12 times) in the microplates. The plates were incubated for 90 min at RT and then washed with buffer 1. 20 ?L of a diluted anti-mouse total IgG peroxidase conjugate (Jackson Inc) was added in each well (1/2500). After 90 min incubation at RT, the plates were washed with buffer 1. The reaction was developed by adding 20 ?L of a tetramethylbenzidine substrate solution in each well. The reaction was chemically stopped after 30 min at RT with HCl (1N (normality)) and absorbance was measured at 450-650 nm on a spectrophotometer (Synergy HTX, Biotek). The results were analyzed in Softmax Pro software using a standard curve and expressed in arbitrary ELISA units by the reciprocal dilution corresponding to an OD of 1.

[1177] As shown in FIG. 28, various MOMP B cell constructs elicited high titers of IgGs which bound to EBs of serovar E at levels similar to the recombinant MOMP. mRNA constructs comprising a transmembrane domain, as well as mRNA coding full-length MOMP, did not give rise to an anti-EB IgG response, as titers were similar to that of the empty LNP control.

[1178] As shown in FIGS. 29 and 30, similar results were observed with binding to EBs of serovar D and serovar G.

Example 11Immunisation with MOMP mRNA Elicits a Similar Immune Response with Different LNP Formulations

[1179] Mice (female C57BL/6) were immunised with mRNA (all modified) encoding recombinant MOMP protein formulated in either of two LNPs. The following groups were evaluated: [1180] MOMP P3 ssHA1 (P3, encoding protein of SEQ ID NO: 53) at a 2 ?g dose, formulated in LNP GL-HEPES-E3-E12-DS-4-E10 (Lipid D) or LNP OF-02 (Lipid A), [1181] MOMP_VDcomb2-extS_ssHA1_Glycneg, (C2-S-GN, encoding protein of SEQ ID NO: 87) at a 2 ?g or 5 ?g dose, formulated in LNP GL-HEPES-E3-E12-DS-4-E10 (Lipid D) or LNP OF-02 (Lipid A).

[1182] Mice received two immunisations of mRNA constructs by IM route, at 0 and 3 weeks (W0 and W3). W3 is alternatively referred to as day 21 herein. The study included empty LNP controls. Blood samples were collected at 0 weeks (W0) and final blood samples and spleens were collected one or two weeks after the last vaccine administration. The cellular response was evaluated in samples collected one week post vaccination with a 2 ?g dose and the humoral response was evaluated in samples collected two weeks post vaccination with a 5 ?g dose. The T specific cellular response was assessed by Intra cellular cell staining (ICCS), as described in Example 7. Induction of specific IgG against recombinant MOMP protein and IgGs binding to EBs of serovars E was assessed as described in Example 8.

MOMP T Cell Construct Elicits a Strong T Cell Response when Formulated in Different LNPs

[1183] Immunisation with MOMP_P3_ssHA1 (P3) gave rise to a significant CD4+ IFN? T cell response, but very low to no CD8+ IFN? T cell response was observed, as shown in FIGS. 31A and 31B, respectively. The level of the response was similar between the two formulations evaluated with P3. In contrast, immunisation with the B cell construct (C2-S-GN) gave rise to both a CD4+ IFN? T cell response and a CD8+ IFN? T cell response.

[1184] Constructs were also assessed for their ability to induce polyfunctional cells. FIG. 31C shows that the CD4+ IFN?+IL2+TNF?+ cell population was induced by P3 in both formulations, at similar levels, as well as by the B cell construct.

MOMP B Cell Construct Induces Specific IgG Against rMOMP and Against EBs of Serovar E when Formulated in Different LNPs

[1185] The MOMP B cell construct (C2-S-GN) gave rise to high titers of IgGs which were specific against recombinant MOMP of serovar E (FIG. 32A) and EBs of serovar E (FIG. 32B) when formulated in different LNPs. Similar levels of IgG were observed between the two formulations.

Example 12Immunisation with a Multivalent mRNA Composition Elicits an Immune Response

[1186] Mice (female C57BL/6) were immunised with a multivalent composition composed of four mRNAs (all modified) encoding the following recombinant proteins: [1187] MOMP P3 ssHA1, (P3, encoding protein of SEQ ID NO: 53), [1188] MOMP_VDcomb2-extS_ssHA1_Glycneg, (C2-S-GN, encoding protein of SEQ ID NO: 87), [1189] CT443_ssHA1 (CT443, encoding protein of SEQ ID NO: 105), and [1190] CT584_ssHA1_GlycNeg (CT584, encoding protein of SEQ ID NO: 108).

[1191] The ratio of the mRNAs was 1:1:1:1. Three doses were evaluated. Specifically, doses of 0.4 ?g, 1.2 ?g and 3.6 ?g of each mRNA were evaluated, corresponding to a total mRNA dose of 1.6 ?g, 4.8 ?g, and 14.4 ?g, respectively for each immunisation. The mRNA composition was formulated in LNP GL-HEPES-E3-E12-DS-4-E10 (Lipid D) or LNP IM-001 (Lipid G). The study included empty LNPs equivalent to the amount present for the two highest mRNA doses, as controls. The immunisation and sampling protocols were as described in Example 11. The T specific cellular response was assessed by Intra cellular cell staining (ICCS), as described in Example 7. Evaluation of the induction of specific IgG against recombinant MOMP protein and IgGs binding to EBs of serovars E was performed as described in Example 8. Little to no reactogenicity was observed at these doses.

Multivalent Composition Elicits CD4+ IFN?+, CD8+ IFN?+, and Polyfunctional Cell Responses (ICCS) when Formulated in Different LNPs

[1192] The multivalent composition was tested for its ability to elicit MOMP-specific CD4+ IFN?+ cells, CD8+ IFN?+ T cells, and polyfunctional cells in mice. As shown in FIG. 33A, the composition gave rise to a significant CD4+ IFN? T cell response that was distinguishable from the empty LNP control. For the multivalent composition, the CD4+ IFN?+IL2+TNF?+ cell population formed the majority, relative to the other cell populations (see FIG. 33B). Little to no IL-17 or IL-10 was observed for any of the tested constructs (data not shown).

[1193] The multivalent composition elicited low levels CD8+ IFN? (see FIG. 34A), with the CD8+ IFN?+IL2-TNF?+ cell population forming the majority, relative to the other cell populations (see FIG. 34B). No difference in the response could be observed between the various doses of mRNA formulated in Lipid D. A difference between the highest dose and the two lower doses was observed for the mRNA formulated in Lipid G.

Multivalent Composition Induces Specific IgG Against rMOMP and Against EBs of Serovar E when

[1194] Formulated in Different LNPs As shown in FIG. 35A, the multivalent composition gave rise to high titers of IgGs which were specific against recombinant MOMP of serovar E at all doses and with both formulations. A dose effect was observed.

[1195] The multivalent composition also gave rise to high titers of specific IgGs which bound to EBs of serovar E at all doses and with both formulations, as shown in FIG. 35B.

Non-MOMP Ct Antigen Constructs Present in the Multivalent Composition are Able to Induce Specific IgG

[1196] Against their Encoded Proteins CT443 and CT584 constructs present in the multivalent composition induce high levels of specific IgG against recombinant CT443 and CT584, respectively, as shown in panels A (CT443) and B (CT584) of FIG. 36. Results were similar between the two formulations tested. Results suggest that interference among antigens did not occur, as high levels of specific IgG were induced against each recombinant protein.

Example 13Immunisation with a Multivalent mRNA Composition Elicits Antibody Binding to Elementary Bodies (EBs) of Serovar E and Serovar G and Respective Recombinant Proteins

[1197] Mice (female C57BL/6) were immunised with monovalent or multivalent mRNAs (all modified) encoding the following recombinant proteins: [1198] MOMP P3 ssHA1, (P3, encoding protein of SEQ ID NO: 53), [1199] MOMP_VDcomb2-extS_ssHA1_Glycneg, (C2-S-GN, encoding protein of SEQ ID NO: 87), [1200] CT443_ssHA1 (CT443, encoding protein of SEQ ID NO: 105), and [1201] CT584_ssHA1_GlycNeg (CT584, encoding protein of SEQ ID NO: 108), [1202] a multivalent composition including the mRNAs encoding MOMP P3, MOMP C2-S-GN, CT443 and CT584-GN described above.

[1203] Mice received two immunisations of mRNA constructs at 1.2 ?g dose, formulated in LNP GL-HEPES-E3-E12-DS-4-E10 (Lipid D) or LNP IM-001 (Lipid G), given by IM route, at 0 and 3 weeks (W0 and W3). The multivalent was formulated with a ratio of 1:1:1:1 of each mRNA construct and injected at 1.2 ?g dose of each mRNA construct, 4.8 ?g of total mRNA. PBS buffer was administered as a control. Blood samples were collected at 0 weeks (W0) and one week after the last vaccine administration to determine seroconversion, which can be detected at this time point. Seroconversion was determined by measuring total IgG from individual sera using automated 384 ELISA as described above in Example 8 for non-MOMP antigen constructs and Example 9 for MOMP antigen constructs. IgG titers were analyzed using one-way ANOVA with group as fixed factor. A Dunnett adjustment was performed.

[1204] As shown in FIGS. 37 and 38, all monovalent and multivalent constructs elicited moderate to high titers of IgGs which were specific against EBs of serovar E and G except the monovalent MOMP_P3. MOMP_P3 mRNA construct did not give rise to an anti-EB IgG response, as titers were similar to that of the buffer control. Similarly, as shown in FIG. 39, all monovalent and multivalent constructs elicited moderate to high titers of IgGs which were specific against their respective recombinant protein except the monovalent MOMP P3.

Example 14Synthesis of IM-001 According to Scheme 2

[1205] ##STR00024##

[1206] Abbreviations: DCM: Dichloromethane, DIPEA: N,N-Diisopropylethylamine, DMAP: 4-Dimethylaminopyridine, EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, EtOAc: Ethyl acetate, NaHCO.sub.3: Sodium hydrogencarbonate, Py: Pyridine, Na.sub.2SO.sub.4: Sodium Sulfate, TEA: Triethylamine, TFA: Trifluoroacetic Acid, MS: Mass spectrometry, ESI-MS: Electrospray ionization mass spectrometry, TLC: Thin Layer Chromatography

Step 1: Synthesis of Intermediate (3)

[1207] ##STR00025##

[1208] As depicted in Scheme 2: To a solution of acid (2) (1.2 g, 1.71 mmol) and isosorbide (1) (0.100 g, 0.68 mmol) in dichloromethane (10 mL) were added DIPEA (0.95 mL, 5.47 mmol), DMAP (0.084 g, 0.68 mmol) and EDC (0.393 g, 2.05 mmol). The resulting mixture was stirred at room temperature for overnight. After 16 h, MS and TLC (30% EtOAc in hexanes) analysis indicated completion of the reaction. The reaction mixture was diluted with dichloromethane and washed with saturated NaHCO.sub.3 solution, water and brine solution. The organic layer was dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The crude residue was purified, and the desired product was eluted at 6% EtOAc in hexanes. The product containing fractions were concentrated to obtain 0.72 g (69%) of pure product.

Results:

[1209] ESI-MS: Calculated C.sub.86H.sub.177N.sub.2O.sub.10Si.sub.4, [M+H.sup.+]=1510.25, Observed=1510.3 and 755.4 [M/2+H.sup.+]

Step 2: Synthesis of IM-001

[1210] ##STR00026##

[1211] As depicted in Scheme 2: To a solution of Intermediate (3) (0.72 g, 0.476 mmol) in tetrahydrofuran (4 mL) was added hydrogen fluoride (70% HF.py complex, 2 mL, 14.298 mmol) at 0? C. and stirred at the same temperature for 5 minutes. Then reaction mixture was warmed to room temperature and stirred for 16 h. MS analysis indicated completion of the reaction. The reaction mixture was diluted with ethyl acetate, quenched by slow addition of solid NaHCO.sub.3 at 0? C., followed by saturated NaHCO.sub.3 solution. The organic layer was washed with sat. NaHCO.sub.3 solution, water and brine. Then dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The crude residue was purified, and the desired product was eluted at 65% EtOAc in hexanes. The purest fractions were concentrated to obtain 0.120 g (24%) of pure product.

Results:

[1212] .sup.1H NMR (400 MHz, CDCl.sub.3) ? 5.30-5.00 (m, 2H), 4.97-4.68 (m, 2H), 4.55-3.71 (m, 8H), 3.57-2.92 (m, 8H), 2.84-2.04 (m, 8H), 1.99-1.01 (m, 76H), 0.88 (t, J=6.8 Hz, 12H).

[1213] ESI-MS: Calculated C.sub.62H.sub.121N.sub.2O.sub.10, [M+H.sup.+]=1053.90, Observed=1053.2 and 527.3 [M/2+H.sup.+]

[1214] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

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Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J Immunol. 138(2):575-81 (PMID: 3540122) [1232] [18] Zhang Y X, et al. (1987). Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J Immunol. 138(2):575-81 (PMID: 3540122) [1233] [19] Cotter T W, et al. (1995). Protective efficacy of major outer membrane protein-specific immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection. Infect Immun. 63(12):4704-14. (PMID: 7591126) [1234] [20] Cohen C R, et al. (2005). Immunoepidemiologic profile of Chlamydia trachomatis infection: importance of heat-shock protein 60 and interferon-gamma. J Infect Dis. 192(4):591-9. (PMID: 16028127) [1235] [21] Brunham R C, et al. (1996). The epidemiology of Chlamydia trachomatis within a sexually transmitted diseases core group. J Infect Dis. 173(4):950-6. 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Antibodies to Variable Domain 4 Linear Epitopes of the Chlamydia trachomatis Major Outer Membrane Protein Are Not Associated with Chlamydia Resolution or Reinfection in Women. mSphere. 5(5). (PMID: 32968007) [1237] [23] Morrison S G, et al. (2005). A predominant role for antibody in acquired immunity to Chlamydial genital tract reinfection. J Immunol. 175(11):7536-42. (PMID: 16301662) [1238] [24] Fadel S, et al. (2007). Chlamydia trachomatis OmcB protein is a surface-exposed glycosaminoglycan-dependent adhesin. J Med Microbiol. 56(Pt 1):15-22. (PMID: 17172511) [1239] [25] Finco O, et al. (2011). Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines. Proc Natl Acad Sci USA. 108(24):9969-74. (PMID: 21628568) [1240] [26] Qi M, et al. (2011). A Chlamydia trachomatis OmcB C-terminal fragment is released into the host cell cytoplasm and is immunogenic in humans. Infect Immun. 79(6):2193-203. (PMID: 21422182) [1241] [27] Olsen A W, et al. (2010). Protection against Chlamydia promoted by a subunit vaccine (CTH1) compared with a primary intranasal infection in a mouse genital challenge model. PLoS One. 5(5). (PMID: 20505822) [1242] [28] Olsen A W, et al. (2014). Characterization of protective immune responses promoted by human antigen targets in a urogenital Chlamydia trachomatis mouse model. Vaccine. 32(6):685-92. (PMID: 24365515) [1243] [29] Markham A P, et al. (2009). Biophysical characterization of Chlamydia trachomatis CT584 supports its potential role as a type III secretion needle tip protein. Biochemistry. 48(43):10353-61. (PMID: 19769366) [1244] [30] Markham A P, et al. (2009). Biophysical characterization of Chlamydia trachomatis CT584 supports its potential role as a type III secretion needle tip protein. Biochemistry. 48(43):10353-61. (PMID: 19769366) [1245] [31] Paes W, et al. (2016). Recombinant polymorphic membrane protein D in combination with a novel, second-generation lipid adjuvant protects against intra-vaginal Chlamydia trachomatis infection in mice. Vaccine. 34(35):4123-4131. (PMID: 27389169) [1246] [32] Paes W, et al. (2018). The Chlamydia trachomatis PmpD adhesin forms higher order structures through disulphide-mediated covalent interactions. PloS one, 13(6), e0198662. (PMID: 29912892)

Sequences

[1247]

TABLE-US-00015 TABLE 10 Protein sequences SEQ nt SEQ ID NO name ID NO(*) other name Full Length constructs 21 MOMP_serD_FL_ssHA1 879 294 22 MOMP_serD_FL_ssHA2 301 302 23 MOMP_serD_FL_ssHA1_C2S 295 296 24 MOMP_serD_FL_ssHA2_C2S 303 304 25 MOMP_serD_FL_ssHA1_Glycneg 298 297 26 MOMP_serD_FL_ssHA2_Glycneg 305 306 27 MOMP_serD_FL_ssHA1_C2S_Glycneg 873 300 28 MOMP_serD_FL_ssHA2_C2S_Glycneg 307 308 29 MOMP_serE_FL_ssHA1 875 309 30 MOMP_serE_FL_ssHA2 317 318 31 MOMP_serE_FL_ssHA1_C2S 311 312 32 MOMP_serE_FL_ssHA2_C2S 319 320 33 MOMP_serE_FL_ssHA1_Glycneg 314 313 34 MOMP_serE_FL_ssHA2_Glycneg 321 322 35 MOMP_serE_FL_ssHA1_C2S_Glycneg 876 316 36 MOMP_serE_FL_ssHA2_C2S_Glycneg 323 324 37 MOMP_serF_FL_ssHA1 880 326 38 MOMP_serF_FL_ssHA2 333 334 39 MOMP_serF_FL_ssHA1_C2S 327 328 40 MOMP_serF_FL_ssHA2_C2S 335 336 41 MOMP_serF_FL_ssHA1_Glycneg 329 330 42 MOMP_serF_FL_ssHA2_Glycneg 338 337 43 MOMP_serF_FL_ssHA1_C2S_Glycneg 331 332 44 MOMP_serF_FL_ssHA2_C2S_Glycneg 339 340 45 MOMP_serG_FL_ssHA1 881 342 46 MOMP_serG_FL_ssHA2 349 350 47 MOMP_serG_FL_ssHA1_C2S 343 874 48 MOMP_serG_FL_ssHA2_C2S 351 352 49 MOMP_serG_FL_ssHA1_Glycneg 345 346 50 MOMP_serG_FL_ssHA2_Glycneg 353 354 51 MOMP_serG_FL_ssHA1_C2S_Glycneg 347 348 52 MOMP_serG_FL_ssHA2_C2S_Glycneg 355 356 T cell constructs 53 MOMP_P3_ssHA1 213 214 54 MOMP_P3_ssHA2 215 216 55 MOMP_P3_ssHA1_C2S 218 217 56 MOMP_P3_ssHA2_C2S 219 220 57 MOMP_P5_ssHA1 222 221 58 MOMP_P5_ssHA2 223 224 59 MOMP_P5_ssHA1_C2S 225 226 60 MOMP_P5_ssHA2_C2S 227 228 VD constructs 61 MOMP_VDcomb1-extP_ssHA1 863 229 62 MOMP_VDcomb1-extP_ssHA2 237 238 63 MOMP_VDcomb1-extP_ssHA1_Glycneg 877 232 64 MOMP_VDcomb1-extP_ssHA2_Glycneg 239 240 65 MOMP_VDcomb1-extP_ssHA1_TMB1 233 234 66 MOMP_VDcomb1-extP_ssHA2_TMB2 241 242 67 MOMP_VDcomb1-extP_ssHA1_TMB1_Glycneg 865 235 68 MOMP_VDcomb1-extP_ssHA2_TMB2_Glycneg 243 244 69 MOMP_VDcomb1-extS_ssHA1 864 245 70 MOMP_VDcomb1-extS_ssHA2 253 254 71 MOMP_VDcomb1-extS_ssHA1_Glycneg 878 248 72 MOMP_VDcomb1-extS_ssHA2_Glycneg 255 256 73 MOMP_VDcomb1-extS_ssHA1_TMB1 249 250 74 MOMP_VDcomb1-extS_ssHA2_TMB2 257 258 75 MOMP_VDcomb1-extS_ssHA1_TMB1_Glycneg 866 251 76 MOMP_VDcomb1-extS_ssHA2_TMB2_Glycneg 259 260 77 MOMP_VDcomb2-extP_ssHA1 261 262 78 MOMP_VDcomb2-extP_ssHA2 269 270 79 MOMP_VDcomb2-extP_ssHA1_Glycneg 868 264 80 MOMP_VDcomb2-extP_ssHA2_Glycneg 869 272 81 MOMP_VDcomb2-extP_ssHA1_TMB1 265 266 82 MOMP_VDcomb2-extP_ssHA2_TMB2 273 274 83 MOMP_VDcomb2-extP_ssHA1_TMB1_Glycneg 871 268 84 MOMP_VDcomb2-extP_ssHA2_TMB2_Glycneg 867 276 85 MOMP_VDcomb2-extS_ssHA1 277 278 86 MOMP_VDcomb2-extS_ssHA2 285 286 87 MOMP_VDcomb2-extS_ssHA1_Glycneg 870 280 88 MOMP_VDcomb2-extS_ssHA2_Glycneg 287 288 89 MOMP_VDcomb2-extS_ssHA1_TMB1 872 282 90 MOMP_VDcomb2-extS_ssHA2_TMB2 289 290 91 MOMP_VDcomb2-extS_ssHA1_TMB1_Glycneg 283 284 92 MOMP_VDcomb2-extS_ssHA2_TMB2_Glycneg 291 292 BENCHMARK constructs 93 Hirep2_ssHA1 862 202 94 Hirep2_ssHA1_TMB1 203 204 95 Hirep2_ssHA1_Glycneg 205 206 96 Hirep2_ssHA1_TMB1_Glycneg 207 208 97 CTH522_ssHA1 209 210 98 CTH522_ssHA1_Glycneg 211 212 99 Hirep2_patent_ssHA1 357 358 100 Hirep2_patent_ssHA1_TMB1 359 360 101 Hirep2_patent_ssHA1_Glycneg 361 362 102 Hirep2_patent_ssHA1_TMB1_Glycneg 363 364 103 CTH522_patent_ssHA1 366 365 104 CTH522_patent_ssHA1_Glycneg 367 368 CT443 105 CT443_ssHA1 369 370 106 CT443_ssHA1_Glycneg 371 372 CT584 107 CT584_ssHA1 374 373 108 CT584_ssHA1_Glycneg 377 378 109 CT584_ssHA1_C2S 376 375 110 CT584_ssHA1_Glycneg_C2S 379 380 CT600 111 CT600_trunc_ssHA1 381 382 112 CT600_trunc_ssHA1_Glycneg 383 384 CT812 113 CT812_pass-domain_ssHA1 385 386 114 CT812_pass-domain_ssHA1_C2S 388 387 115 CT812_ext-pass-domain_ssHA1 389 390 116 CT812_ext-pass-domain_ssHA1_C2S 391 392 117 CT812_pass-domain_ssHA1_HPX1- 399 400 1_HPX2-1 118 CT812_pass-domain_ssHA1_HPX1- 411 412 1_HPX2-3 119 CT812_pass-domain_ssHA1_HPX1- 393 394 2_HPX2-1 120 CT812_pass-domain_ssHA1_HPX1- 405 406 2_HPX2-3 121 CT812_pass-domain_ssHA1_HPX1- 413 414 1_HPX2-1_C2S 122 CT812_pass-domain_ssHA1_HPX1- 417 418 1_HPX2-3_C2S 123 CT812_pass-domain_ssHA1_HPX1- 395 396 2_HPX2-1_C2S 124 CT812_pass-domain_ssHA1_HPX1- 407 408 2_HPX2-3_C2S 125 CT812_ext-pass-domain_ssHA1_HPX1- 403 404 1_HPX2-1 126 CT812_ext-pass-domain_ssHA1_HPX1- 401 402 1_HPX2-3 127 CT812_ext-pass-domain_ssHA1_HPX1- 421 422 2_HPX2-1 128 CT812_ext-pass-domain_ssHA1_HPX1- 419 420 2_HPX2-3 129 CT812_ext-pass-domain_ssHA1_HPX1- 423 424 1_HPX2-1_C2S 130 CT812_ext-pass-domain_ssHA1_HPX1- 415 416 1_HPX2-3_C2S 131 CT812_ext-pass-domain_ssHA1_HPX1- 409 410 2_HPX2-1_C2S 132 CT812_ext-pass-domain_ssHA1_HPX1- 397 398 2_HPX2-3_C2S T cell MOMP protein 133 MOMP_P5_6his_coli Full Length MOMP protein 134 MOMP_SerE_FL_Wt 135 MOMP_serE_FL_6his_coli 136 MOMP_serE_FL_C2S_6his_coli BENCHMARK constructs protein 142 rec SSI CTH522_6his_coli Non-MOMP constructs 143 CT443_6his_coli 538 144 CT443NterTrunc_6his_coli 145 6his_CT584_coli 536 146 6his_CT600_coli 537 147 CT812_ext-pass-domain_6his_coli 197 rCT812 198 rCT443 Pilot constructs 148 MC3 MOMP or MC3 SER E MOMP 425 OmpA Serovar E 149 MC3 MOMP (human leader) or MC3 SER 426 OmpA Serovar E w/Human E MOMP human leader Leader 150 OmpA Serovar E w/Human Leader 151 OF-02 MOMP or OF-02 SER E MOMP 425 OmpA Serovar E 152 OF-02 MOMP (human leader) or OF-02 426 OmpA Serovar E w/Human SER E MOMP human leader Leader 153 427 OmpA Serovar D w/Human Leader 154 mRNA MOMP WT 425 OmpA Serovar E 155 mRNA MNR MOMP E FL MRT12514 861 MNR CT-681 (wild type)- no tag 156 mRNA MOMP ?VD4 428 MOMP/D-VD4 157 mRNA MOMP ?VD24 or mRNA 429 MOMP/D-VD2_VD4 UNR MOMPE DII:IV 158 mRNA MOMP ?VD24 430 MOMP/D-VD1_VD2_VD4 159 mRNA MOMP ?VD1234 431 MOMP/D-VD3_VD1_VD2_VD4 160 mRNA MOMP ?signal peptide 432 MOMP/D-signalpep 161 mRNA MOMP ?VD124 452 MOMP:DVD1 162 mRNA MOMP ?VD2 453 MOMP:DVD2 163 mRNA MOMP ?VD3 454 MOMP:DVD3 164 mRNA MOMP ?VD23 455 MOMP:DVD2:DVD3 165 mRNA MOMP ?VD123 456 MOMP:DVD1:DVD2:DVD3 166 mRNA MOMP ?VD234 457 MOMP:DVD2:DVD3:DVD4 167 mRNA MOMP ?VD134 458 MOMP:DVD1:DVD3:DVD4 168 CT812: 1-1530 433 CT812: 1-1530 169 CT812: 1-761 434 CT812: 1-761 170 Nan96-SS:CT812: 32-761 or CT812 435 Nan96-SS:CT812: 32-761 171 CT600: 1-188 436 CT600: 1-188 172 Nan96-SS:CT600: 2-188 or CT600 437 Nan96HA-SS:CT600: 2-188 173 CT443: 1-576 438 CT443: 1-576 174 Nan96-SS:CT443: 32-576 or CT443 439 Nan96HA-SS:CT443: 32-576 175 CT584: 1-183 440 CT584: 1-183 176 CT584: 1-183* 441 CT584: 1-183* 177 Nan96HA-SS:CT584: 2-183 or CT584 442 Nan96HA-SS:CT584: 2-183 BENCHMARK constructs 506 Hirep2 841 Hirep2_patent 695 696 842 CTH522 843 CTH522_patent (*)for protein sequences where two corresponding nucleic acid sequences are mentioned, the first sequence was tested in mice (where applicable)

TABLE-US-00016 TABLE 11 Sequence information SEQ ID NO: Name P3 and P5 VD loops 462 VD1_P3 463 VD2_P3 464 VD3_P3 465 VD4_P3 466 VD1_P5 467 VD2_P5 468 VD3_P5 469 VD4_P5 MOMP proteins without SS 470 MOMP_serD_FL 471 MOMP_serD_FL_C2S 472 MOMP_serD_FL_Glycneg 473 MOMP_serD_FL_C2S_Glycneg 474 MOMP_serE_FL 475 MOMP_serE_FL_C2S 476 MOMP_serE_FL_Glycneg 477 MOMP_serE_FL_C2S_Glycneg 478 MOMP_serF_FL 479 MOMP_serF_FL_C2S 480 MOMP_serF_FL_Glycneg 481 MOMP_serF_FL_C2S_Glycneg 482 MOMP_serG_FL 483 MOMP_serG_FL_C2S 484 MOMP_serG_FL_Glycneg 485 MOMP_serG_FL_C2S_Glycneg T cell constructs 486 MOMP_P3 487 MOMP_P3_C2S 488 MOMP_P5 489 MOMP_P5_C2S VD constructs 490 MOMP_VDcomb1-extP 491 MOMP_VDcomb1-extP_Glycneg 492 MOMP_VDcomb1-extP_TMB1 493 MOMP_VDcomb1-extP_TMB1_Glycneg 494 MOMP_VDcomb1-extS 495 MOMP_VDcomb1-extS_Glycneg 496 MOMP_VDcomb1-extS_TMB1 499 MOMP_VDcomb2-extP_Glycneg 500 MOMP_VDcomb2-extP_TMB1 501 MOMP_VDcomb2-extP_TMB1_Glycneg 502 MOMP_VDcomb2-extS 503 MOMP_VDcomb2-extS_Glycneg 504 MOMP_VDcomb2-extS_TMB1 505 MOMP_VDcomb2-extS_TMB1_Glycneg non-MOMP proteins without SS 507 CT443 508 CT443_Glycneg CT584 509 CT584 510 CT584_Glycneg 511 CT584_C2S 512 CT584_Glycneg_C2S CT600 844 CT600_ref 513 CT600_trunc 514 CT600_trunc_Glycneg CT812 515 CT812_ref 516 CT812_pass-domain 517 CT812_pass-domain_C2S 518 CT812_ext-pass-domain 519 CT812_ext-pass-domain_C2S 520 CT812_pass-domain_HPX1-1_HPX2-1 521 CT812_pass-domain_HPX1-1_HPX2-3 522 CT812_pass-domain_HPX1-2_HPX2-1 523 CT812_pass-domain_HPX1-2_HPX2-3 524 CT812_pass-domain_HPX1-1_HPX2-1_C2S 525 CT812_pass-domain_HPX1-1_HPX2-3_C2S 526 CT812_pass-domain_HPX1-2_HPX2-1_C2S 527 CT812_pass-domain_HPX1-2_HPX2-3_C2S 528 CT812_ext-pass-domain_HPX1-1_HPX2-1 529 CT812_ext-pass-domain_HPX1-1_HPX2-3 530 CT812_ext-pass-domain_HPX1-2_HPX2-1 531 CT812_ext-pass-domain_HPX1-2_HPX2-3 532 CT812_ext-pass-domain_HPX1-1_HPX2-1_C2S 533 CT812_ext-pass-domain_HPX1-1_HPX2-3_C2S

TABLE-US-00017 TABLE 12 Nucleic acid sequences Corresp. SEQ SEQ ID NO. ID NO: Construct name (with SS) 539 Hirep2_ssHA1 862 540 Hirep2_ssHA1 202 541 Hirep2_ssHA1_TMB1 203 542 Hirep2_ssHA1_TMB1 204 543 Hirep2_ssHA1_Glycneg 205 544 Hirep2_ssHA1_Glycneg 206 545 Hirep2_ssHA1_TMB1_Glycneg 207 546 Hirep2_ssHA1_TMB1_Glycneg 208 547 CTH522_ssHA1 209 548 CTH522_ssHA1 210 549 CTH522_ssHA1_Glycneg 211 550 CTH522_ssHA1_Glycneg 212 551 MOMP_P3_ssHA1 213 552 MOMP_P3_ssHA1 214 553 MOMP_P3_ssHA2 215 554 MOMP_P3_ssHA2 216 555 MOMP_P3_ssHA1_C2S 217 556 MOMP_P3_ssHA1_C2S 218 557 MOMP_P3_ssHA2_C2S 219 558 MOMP_P3_ssHA2_C2S 220 559 MOMP_P5_ssHA1 221 560 MOMP_P5_ssHA1 222 561 MOMP_P5_ssHA2 223 562 MOMP_P5_ssHA2 224 563 MOMP_P5_ssHA1_C2S 225 564 MOMP_P5_ssHA1_C2S 226 565 MOMP_P5_ssHA2_C2S 227 566 MOMP_P5_ssHA2_C2S 228 567 MOMP_VDcomb1-extP_ssHA1 229 568 MOMP_VDcomb1-extP_ssHA1 863 569 MOMP_VDcomb1-extP_ssHA1_Glycneg 877 570 MOMP_VDcomb1-extP_ssHA1_Glycneg 232 571 MOMP_VDcomb1-extP_ssHA1_TMB1 233 572 MOMP_VDcomb1-extP_ssHA1_TMB1 234 573 MOMP_VDcomb1-extP_ssHA1_TMB1_Glycneg 235 574 MOMP_VDcomb1-extP_ssHA1_TMB1_Glycneg 865 575 MOMP_VDcomb1-extP_ssHA2 237 576 MOMP_VDcomb1-extP_ssHA2 238 577 MOMP_VDcomb1-extP_ssHA2_Glycneg 239 578 MOMP_VDcomb1-extP_ssHA2_Glycneg 240 579 MOMP_VDcomb1-extP_ssHA2_TMB2 241 580 MOMP_VDcomb1-extP_ssHA2_TMB2 242 581 MOMP_VDcomb1-extP_ssHA2_TMB2_Glycneg 243 582 MOMP_VDcomb1-extP_ssHA2_TMB2_Glycneg 244 583 MOMP_VD-comb1-extS_ssHA1 245 584 MOMP_VD-comb1-extS_ssHA1 864 585 MOMP_VD-comb1-extS_ssHA1_Glycneg 878 586 MOMP_VD-comb1-extS_ssHA1_Glycneg 248 587 MOMP_VD-comb1-extS_ssHA1_TMB1 249 588 MOMP_VD-comb1-extS_ssHA1_TMB1 250 589 MOMP_VD-comb1-extS_ssHA1_TMB1_Glycneg 251 590 MOMP_VD-comb1-extS_ssHA1_TMB1_Glycneg 866 591 MOMP_VD-comb1-extS_ssHA2 253 592 MOMP_VD-comb1-extS_ssHA2 254 593 MOMP_VD-comb1-extS_ssHA2_Glycneg 255 594 MOMP_VD-comb1-extS_ssHA2_Glycneg 256 595 MOMP_VD-comb1-extS_ssHA2_TMB2 257 596 MOMP_VD-comb1-extS_ssHA2_TMB2 258 597 MOMP_VD-comb1-extS_ssHA2_TMB2_Glycneg 259 598 MOMP_VD-comb1-extS_ssHA2_TMB2_Glycneg 260 599 MOMP_VDcomb2-extP_ssHA1 261 600 MOMP_VDcomb2-extP_ssHA1 262 601 MOMP_VDcomb2-extP_ssHA1_Glycneg 868 602 MOMP_VDcomb2-extP_ssHA1_Glycneg 264 603 MOMP_VDcomb2-extP_ssHA1_TMB1 265 604 MOMP_VDcomb2-extP_ssHA1_TMB1 266 605 MOMP_VDcomb2-extP_ssHA1_TMB1_Glycneg 871 606 MOMP_VDcomb2-extP_ssHA1_TMB1_Glycneg 268 607 MOMP_VDcomb2-extP_ssHA2 269 608 MOMP_VDcomb2-extP_ssHA2 270 609 MOMP_VDcomb2-extP_ssHA2_Glycneg 869 610 MOMP_VDcomb2-extP_ssHA2_Glycneg 272 611 MOMP_VDcomb2-extP_ssHA2_TMB2 273 612 MOMP_VDcomb2-extP_ssHA2_TMB2 274 613 MOMP_VDcomb2-extP_ssHA2_TMB2_Glycneg 867 614 MOMP_VDcomb2-extP_ssHA2_TMB2_Glycneg 276 615 MOMP_VDcomb2-extS_ssHA1 277 616 MOMP_VDcomb2-extS_ssHA1 278 617 MOMP_VDcomb2-extS_ssHA1_Glycneg 870 618 MOMP_VDcomb2-extS_ssHA1_Glycneg 280 619 MOMP_VDcomb2-extS_ssHA1_TMB1 872 620 MOMP_VDcomb2-extS_ssHA1_TMB1 282 621 MOMP_VDcomb2-extS_ssHA1_TMB1_Glycneg 283 622 MOMP_VDcomb2-extS_ssHA1_TMB1_Glycneg 284 623 MOMP_VDcomb2-extS_ssHA2 285 624 MOMP_VDcomb2-extS_ssHA2 286 625 MOMP_VDcomb2-extS_ssHA2_Glycneg 287 626 MOMP_VDcomb2-extS_ssHA2_Glycneg 288 627 MOMP_VDcomb2-extS_ssHA2_TMB2 289 628 MOMP_VDcomb2-extS_ssHA2_TMB2 290 629 MOMP_VDcomb2-extS_ssHA2_TMB2_Glycneg 291 630 MOMP_VDcomb2-extS_ssHA2_TMB2_Glycneg 292 631 MOMP_serD_FL_ssHA1 879 632 MOMP_serD_FL_ssHA1 294 633 MOMP_serD_FL_ssHA1_C2S 295 634 MOMP_serD_FL_ssHA1_C2S 296 635 MOMP_serD_FL_ssHA1_Glycneg 297 636 MOMP_serD_FL_ssHA1_Glycneg 298 637 MOMP_serD_FL_ssHA1_C2S_Glycneg 873 638 MOMP_serD_FL_ssHA1_C2S_Glycneg 300 639 MOMP_serD_FL_ssHA2 301 640 MOMP_serD_FL_ssHA2 302 641 MOMP_serD_FL_ssHA2_C2S 303 642 MOMP_serD_FL_ssHA2_C2S 304 643 MOMP_serD_FL_ssHA2_Glycneg 305 644 MOMP_serD_FL_ssHA2_Glycneg 306 645 MOMP_serD_FL_ssHA2_C2S_Glycneg 307 646 MOMP_serD_FL_ssHA2_C2S_Glycneg 308 647 MOMP_serE_FL_ssHA1 309 648 MOMP_serE_FL_ssHA1 875 649 MOMP_serE_FL_ssHA1_C2S 311 650 MOMP_serE_FL_ssHA1_C2S 312 651 MOMP_serE_ssHA1_Glycneg 313 652 MOMP_serE_ssHA1_Glycneg 314 653 MOMP_serE_ssHA1_C2S_Glycneg 876 654 MOMP_serE_ssHA1_C2S_Glycneg 316 655 MOMP_serE_FL_ssHA2 317 656 MOMP_serE_FL_ssHA2 318 657 MOMP_serE_FL_ssHA2_C2S 319 658 MOMP_serE_FL_ssHA2_C2S 320 659 MOMP_serE_ssHA2_Glycneg 321 660 MOMP_serE_ssHA2_Glycneg 322 661 MOMP_serE_ssHA2_C2S_Glycneg 323 662 MOMP_serE_ssHA2_C2S_Glycneg 324 663 MOMP_serF_FL_ssHA1 880 664 MOMP_serF_FL_ssHA1 326 665 MOMP_serF_FL_ssHA1_C2S 327 666 MOMP_serF_FL_ssHA1_C2S 328 667 MOMP_serF_FL_ssHA1_Glycneg 329 668 MOMP_serF_FL_ssHA1_Glycneg 330 669 MOMP_serF_FL_ssHA1_C2S_Glycneg 331 670 MOMP_serF_FL_ssHA1_C2S_Glycneg 332 671 MOMP_serF_FL_ssHA2 333 672 MOMP_serF_FL_ssHA2 334 673 MOMP_serF_FL_ssHA2_C2S 335 674 MOMP_serF_FL_ssHA2_C2S 336 675 MOMP_serF_FL_ssHA2_Glycneg 337 676 MOMP_serF_FL_ssHA2_Glycneg 338 677 MOMP_serF_FL_ssHA2_C2S_Glycneg 339 678 MOMP_serF_FL_ssHA2_C2S_Glycneg 340 679 MOMP_serG_FL_ssHA1 881 680 MOMP_serG_FL_ssHA1 342 681 MOMP_serG_FL_ssHA1_C2S 343 682 MOMP_serG_FL_ssHA1_C2S 874 683 MOMP_serG_FL_ssHA1_Glycneg 345 684 MOMP_serG_FL_ssHA1_Glycneg 346 685 MOMP_serG_FL_ssHA1_C2S 347 686 MOMP_serG_FL_ssHA1_C2S 348 687 MOMP_serG_FL_ssHA2 349 688 MOMP_serG_FL_ssHA2 350 689 MOMP_serG_FL_ssHA2_C2S 351 690 MOMP_serG_FL_ssHA2_C2S 352 691 MOMP_serG_FL_ssHA2_Glycneg 353 692 MOMP_serG_FL_ssHA2_Glycneg 354 693 MOMP_serG_FL_ssHA2_C2S 355 694 MOMP_serG_FL_ssHA2_C2S 356 695 Hirep2_patent_ssHA1 [CO1] PolyA 357 696 Hirep2_patent_ssHA1 [CO2] PolyA 358 697 Hirep2_patent_ssHA1_TMB1 [CO1] PolyA 359 698 Hirep2_patent_ssHA1_TMB1 [CO2] PolyA 360 699 Hirep2_patent_ssHA1_Glycneg [CO1] PolyA 361 700 Hirep2_patent_ssHA1_Glycneg [CO2] PolyA 362 701 Hirep2_patent_ssHA1_TMB1_Glycneg [CO1] PolyA 363 702 Hirep2_patent_ssHA1_TMB1_Glycneg [CO2] PolyA 364 703 CTH522_patent_ssHA1 [CO1] PolyA 365 704 CTH522_patent_ssHA1 [CO2] PolyA 366 705 CTH522_patent_ssHA1_Glycneg [CO1] PolyA 367 706 CTH522_patent_ssHA1_Glycneg [CO2] PolyA 368 707 CT443_ssHA1 PolyA 369 708 CT443_ssHA1 PolyA 370 709 CT443_ssHA1_Glycneg PolyA 371 710 CT443_ssHA1_Glycneg PolyA 372 711 CT584_ssHA1 PolyA 373 712 CT584_ssHA1 PolyA 374 713 CT584_ssHA1_C2S PolyA 375 714 CT584_ssHA1_C2S PolyA 376 715 CT584_ssHA1_Glycneg PolyA 377 716 CT584_ssHA1_Glycneg PolyA 378 717 CT584_ssHA1_Glycneg_C2S PolyA 379 718 CT584_ssHA1_Glycneg_C2S PolyA 380 719 CT600_trunc_ssHA1 PolyA 381 720 CT600_trunc_ssHA1 PolyA 382 721 CT600_trunc_ssHA1_Glycneg PolyA 383 722 CT600_trunc_ssHA1_Glycneg PolyA 384 723 CT812_pass-domain_ssHA1 PolyA 385 724 CT812_pass-domain_ssHA1 PolyA 386 725 CT812_pass-domain_ssHA1_C2S PolyA 387 726 CT812_pass-domain_ssHA1_C2S PolyA 388 727 CT812_ext-pass-domain_ssHA1 PolyA 389 728 CT812_ext-pass-domain_ssHA1 PolyA 390 729 CT812_ext-pass-domain_ssHA1_C2S PolyA 391 730 CT812_ext-pass-domain_ssHA1_C2S PolyA 392 731 CT812_pass-domain_ssHA1_HPX1-2_HPX2-1 CO1 393 732 CT812_pass-domain_ssHA1_HPX1-2_HPX2-1 CO2 394 733 CT812_pass-domain_ssHA1_HPX1-2_HPX2-1_C2S CO1 395 734 CT812_pass-domain_ssHA1_HPX1-2_HPX2-1_C2S CO2 396 735 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-3_C2S CO1 397 736 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-3_C2S CO2 398 737 CT812_pass-domain_ssHA1_HPX1-1_HPX2-1 CO1 399 738 CT812_pass-domain_ssHA1_HPX1-1_HPX2-1 CO2 400 739 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-3 CO1 401 740 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-3 CO2 402 741 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-1 CO1 403 742 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-1 CO2 404 743 CT812_pass-domain_ssHA1_HPX1-2_HPX2-3 CO1 405 744 CT812_pass-domain_ssHA1_HPX1-2_HPX2-3 CO2 406 745 CT812_pass-domain_ssHA1_HPX1-2_HPX2-3_C2S CO1 407 746 CT812_pass-domain_ssHA1_HPX1-2_HPX2-3_C2S CO2 408 747 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-1_C2S CO1 409 748 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-1_C2S CO2 410 749 CT812_pass-domain_ssHA1_HPX1-1_HPX2-3 CO1 411 750 CT812_pass-domain_ssHA1_HPX1-1_HPX2-3 CO2 412 751 CT812_pass-domain_ssHA1_HPX1-1_HPX2-1_C2S CO1 413 752 CT812_pass-domain_ssHA1_HPX1-1_HPX2-1_C2S CO2 414 753 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-3_C2S CO1 415 754 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-3_C2S CO2 416 755 CT812_pass-domain_ssHA1_HPX1-1_HPX2-3_C2S CO1 417 756 CT812_pass-domain_ssHA1_HPX1-1_HPX2-3_C2S CO2 418 757 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-3 CO1 419 758 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-3 CO2 420 759 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-1 CO1 421 760 CT812_ext-pass-domain_ssHA1_HPX1-2_HPX2-1 CO2 422 761 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-1_C2S CO1 423 762 CT812_ext-pass-domain_ssHA1_HPX1-1_HPX2-1_C2S CO2 424 763 425 764 426 765 427 766 428 767 429 768 430 769 431 770 432 771 433 772 434 773 435 774 436 775 437 776 438 777 439 778 440 779 441 780 442 790 452 791 453 792 454 793 455 794 456 795 457 796 458 797 861 800 536 801 537 802 538